Novel tricyclic nucleosides or nucleotides as therapeutic agents

ABSTRACT

Nucleosides and nucleotides containing a tricyclic base portion thereof are useful for treating infectious diseases and proliferative disorders, such as viral infections or cancer respectively.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.10/568,917, now pending, which is a U.S. National Stage ofPCT/US2004/027819, accorded an International Filing Date of Aug. 27,2004, which claims the benefit under 35 USC 119(e) of U.S. ProvisionalApplication No. 60/498,425 filed Aug. 27, 2003; all of theseapplications are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to novel tricyclic nucleosides andnucleotides, their preparation, and their use for the treatment ofinfectious disease, including viral infections, and of proliferativedisorders, including cancer.

BACKGROUND OF THE INVENTION

Viral infections are a major threat to human health and account for manyserious infectious diseases. Hepatitis C virus (HCV), a major cause ofviral hepatitis, has infected more than 200 million people worldwide.Current treatment for HCV infection is restricted to immunotherapy withinterferon-α alone or in combination with ribavirin, a nucleosideanalog. This treatment is effective in only about half the patientpopulation. Therefore, there is an urgent need for new HCV drugs.Hepatitis C virus comprises a positive-strand RNA genome enclosed in anucleocapsid and lipid envelope and consists of approximately 9600ribonucleotides, which encodes a polyprotein of about 3000 amino acids(Dymock et al. Antiviral Chemistry & Chemotherapy 2000, 11, 79). A HCVprotein, NS5B, released from the polyprotein, possesses polymeraseactivity and is involved in the synthesis of double-stranded RNA fromthe single-stranded viral RNA genome that serves as the template. Thereproduction of HCV virus may be prevented through the manipulation ofNS5B's polymerase activity. The competitive inhibition of NS5B proteinwould suppress or prevent the formation of the double-stranded HCV RNA.Alternatively, a nucleoside analog also may be incorporated into theextending RNA strand and act as a chain-terminator. Furthermore, adeteriorating nucleoside analog also may be incorporated into theextending RNA, which may cause mutagenic damage to the viral genome.Recently, several PCT patent applications (WO 99/43691, WO 01/32153, WO01/60315, WO 01/79246, WO 01/90121, WO 01/92282, WO 02/18404, WO02/057287, WO 02/057425) have described nucleoside analogs as anti-HCVagents in in vitro assays.

Hepatitis B virus (HBV) has acutely infected almost a third of theworld's human population, and about 5% of the infected are chroniccarriers of the virus (Delaney IV et al. Antiviral Chemistry &Chemotherapy 2001, 12, 1-35). Chronic HBV infection causes liver damagethat frequently progresses to cirrhosis and/or liver cancer later in thelife. Despite the availability and widespread use of effective vaccinesand chemotherapy, the number of chronic carriers approaches 400 millionworldwide. Therefore, more effective anti-HBV drugs need to bedeveloped. Human immunodeficiency virus (HIV) causes progressivedegeneration of the immune system, leading to the development of AIDS. Anumber of drugs have been used clinically, including reversetranscriptase inhibitors and protease inhibitors. Currently, combinationtherapies are used widely for the treatment of AIDS in order to reducethe drug resistance. Despite the progress in the development of anti-HIVdrugs, AIDS is still one of the leading epidemic diseases. Certain acuteviral infections also impose a great threat to human life, including thenewly-discovered West Nile virus and SARS virus.

Bacterial infections long have been the sources of many infectiousdiseases. The widespread use of antibiotics produces many new strains oflife-threatening bacteria Fungal infections are another type ofinfectious diseases, some of which also can be life-threatening. Thereis an increasing demand for the treatment of bacterial and fungalinfections. Antimicrobial drugs based on new mechanisms of action areespecially important.

Proliferative disorders are one of the major life-threatening diseasesand have been intensively investigated for decades. Cancer now is thesecond leading cause of death in the United States, and over 500,000people die annually from this proliferative disorder. All of the variouscells types of the body can be transformed into benign or malignanttumor cells. Transformation of normal cells into cancer cells is acomplex process and thus far is not fully understood. The treatment ofcancer consists of surgery, radiation, and chemotherapy. Whilechemotherapy can be used to treat all types of cancer, surgery andradiation therapy are limited to certain cancer at certain sites of thebody. There are a number of anticancer drugs widely used clinically.Among them are alkylating agent such as cisplatin, antimetabolites, suchas 5-fluorouracil, and gemcitabine. Although surgery, radiation, andchemotherapies are available to treat cancer patients, there is no curefor cancer at the present time. Cancer research is still one of the mostimportant tasks in medical and pharmaceutical organizations.

Nucleoside drugs have been used clinically for the treatment of viralinfections and proliferative disorders for decades. Most of thenucleoside drugs are classified as antimetabolites. After they entercells, nucleoside analogs are phosphorylated successively to nucleoside5′-monophosphates, 5′-diphosphates, and 5′-triphosphates. In most cases,nucleoside triphosphates, e.g., 3′-azido-3′-deoxythymidine (AZT, ananti-HIV drug) triphosphate and arabinosylcytosine (cytarabine, ananticancer drug) triphosphate, are the active chemical entities thatinhibit DNA or RNA synthesis, through a competitive inhibition ofpolymerases and subsequent incorporation of modified nucleotides intoDNA or RNA sequences. In a few cases, nucleoside analogs exert effectsat lower phosphate levels. For instance, 5-fluoro-2′-deoxyuridine (ananticancer drug) 5′-monophosphate and 2′,2′-difluoro-2′-deoxycytidine(an anticancer drug) 5′-diphosphate have been shown to inhibitthymidylate synthase and ribonucleotide reductase, respectively.Although nucleoside analogs themselves may act at the nonphosphate levelsuch as the inhibitors of adenosine kinases and the ligands of adenosinereceptors, currently, clinically-useful nucleoside drugs primarilydepend on cellular activation by nucleoside kinases and nucleotidekinases.

At least, two criteria are pertinent for nucleoside antiviral drugs: 1.nucleoside analogs should anabolize to nucleotides in cells; and 2. theanabolized nucleotides should target selectively viral enzymes. In orderto be phosphorylated in cells and selectively to target preferredenzymes, nucleoside analogs should have favorable modifications on theirsugar and base moieties. To obtain such favorable nucleoside analogs, ageneral approach is to generate diverse nucleoside analogs by modifyingthe base or the sugar, or by modifying both base and sugar moieties.Numerous examples exist in the literature for the synthesis of a varietyof modified nucleosides (Chemistry of Nucleosides and Nucleotides Vol. 1(1988), Vol. 2 (1991), Vol. 3 (1994), edited by Leroy B. Townsend,Plenum Press).

However, there are certain classes of nucleoside compounds that were notexplored intensively for their antiviral and anti-proliferativeactivities before the present invention. A class of such compounds istricyclic nucleosides. Disclosures on tricyclic nucleosides are verylimited considering the existence of various tricyclic heterocycles. Awell-known tricyclic nucleoside is triciribine (TCN), having potentcytotoxicity against cancer cells (Porcari et al. J. Med. Chem. 2000,43, 2438-2448). A number of its modified derivatives were prepared andscreened against viruses and cancer (Porcari et al. NucleosidesNucleotides 1999, 18, 2475-2497; J. Med. Chem. 2000, 43, 2457-2463).Another known tricyclic nucleoside is2-(2-deoxy-β-D-erythro-pentofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenz[cd]azulen-7-one,but its biological activity was not reported (Helv. Chim. Acta, 2000,83, 911-927). The PCT publication WO 03/061385 describes tricyclicnucleoside libraries. The present invention discloses novel tricyclicnucleosides and nucleotides and their use for the treatment ofinfectious disease, including viral infections, and of proliferativedisorders, including cancer.

SUMMARY OF THE INVENTION

The present invention relates to novel tricyclic nucleosides andderivatives thereof, their preparation, and their use for the treatmentof viral infections and proliferative disorders.

In one embodiment, a compound of the formula (I) which may be a D- orL-nucleoside is provided

wherein

A is O, S, CH₂, CHF, or CF₂;

R¹, R², R^(2′), R³, R^(3′), and R⁴ are independently selected from thegroup consisting of H, F, Cl, Br, I, OH, SH, NH₂, NHOH, NHNH₂, N₃, COOH,CN, CONH₂, C(S)NH₂, COOR, R, OR, SR, SSR, NIHR, and NR₂;

R^(4′) is -L-R⁵;

L is selected from the group consisting of O, S, NH, NR, CY₂O, CY₂S,CY₂NH, CY₂, CY₂CY₂, CY₂OCY₂, CY₂SCY₂, and CY₂NHCY₂, wherein Y isselected from the group consisting of H, F, Cl, Br, alkyl, alkenyl, andalkynyl, wherein alkyl, alkenyl, and alkynyl may each optionally containone or more heteroatoms;

R⁵ is OH, monophosphate, diphosphate, or triphosphate, optionally maskedwith prodrug moieties, or a mono di or triphosphate mimic;

B is a base selected from the group of heterocycles consisting of

each Z is independently selected from the group consisting ofN,N-⁻BH₂GM⁺, C-G, O, S, NR, >C═O, >C═S, >C═NH, >C═NR, >S═O, >S(O)₂ andCH-G;

wherein if Z is a participant in a π bond (double bond), Z isindependently N or C-G;

wherein if Z is not a participant in a π bond, Z is independentlyN-⁻BH₂GM⁺, O, S, NR, >C═O, >C═S, >C═NH, >C═NR, >S═O, >S(O)₂ and CH-G;

X is O, S, SO, SO₂, Se, SeO, SeO₂, NH, or NR;

W is C, CH or N;

wherein if W is a participant in one π bond, W is C;

wherein if W is not a participant in a π bond, W is CH or N; and

⁻BH₂GM⁺ is an ion pair and M⁺ is a cation;

G is selected from the group consisting of H, F, Cl, Br, I, OH, SH, NH₂,NHOH, N₃, COOH, CN, CONH₂, C(S)NH₂, C(═NH)NH₂, R, OR, SR, NHR, and NR₂,when two or more G groups are present on a molecule, they may be same asor different from one another;

R is selected from the group consisting of alkyl, alkenyl, alkynyl,aryl, acyl, and aralkyl, optionally containing one or more heteroatoms;

dashed lines (---) indicate a possible π or double bond.

Thus, structures of formulae II and III may have one or more ring doublebonds and, in some instances, may have two or more ring double bonds.

In one preferred embodiment L is CH₂.

Preferably W is C. Preferably X is NH.

In another embodiment, the seven-membered ring portion of the basecontains one or two and preferably one N in the backbone of the ring.

In another embodiment, the Z in the five-membered ring of the base is C.

In yet another embodiment, each Z in the seven-membered ring portion ofthe base is preferably C-G, >C═O, >C═S. Preferably CH-G is CH₂, CH-halo,and C-G is CH, C-alkyl preferably CCH₂, C—OR, preferably, C—O alkyl,more preferably COCH₃.

In one embodiment, at least one of R² or R^(2′) is not H. In anotherpreferred embodiment, the sugar

Thus, compounds of the invention may have the formula

Some embodiments, in compounds of the invention of formula (I), B is abase selected from the group of heterocycles consisting of

Z¹, Z², Z³, Z⁴, Z⁷ and Z⁸ independently N or C-G;

Z⁵, Z⁶, and Z⁹ are independently selected from the group consisting ofN—⁻BH₂GM⁺, O, S, NR, >C═O, >C═S, >C═NH, >C═NR, >S═O, >S(O)₂ and CH-G.

In a preferred embodiment, the base is

Preferably the compound of the invention is not

In another aspect the component is not

In another embodiment, is provided a method for the treatment of a viralor bacterial infection, or proliferative disorder comprisingadministering an effective amount of a compound of the formula (I), or apharmaceutically acceptable salt or a prodrug thereof, optionally incombination with one or more antiviral, antibacterial, orantiproliferative agents. In one aspect, the viral infection is causedby an RNA virus, such as HCV or a DNA or retrovirus such as HBV or HIV.

The invention is also directed to a process of making compounds of theinvention. For example, a compound having the formula

may be made by cyclising a compound having the formula

wherein each of Z¹-Z⁴ is independently Z; and wherein each of W¹-W² isindependently W. Preferably, in this process A is O, CH₂ or optionallyprotected N;

W¹ is C (if p bond) or N (if no p bond);

W² is C, CH or N;

W⁴ is H or trialkyltin;

Z¹ and Z² are each independently CH, C-halogen, C-alkyl, C-aryl,C—O-alkyl or C—S-alkyl;

Z³ is CH, C-alkyl, C-halogen, N, CNHR, CNH₂, CNR₂, C═O, or C═S;

Z⁴ is CH, C-halogen, C-alkyl, C-aryl, C—O-alkyl, C—S-alkyl, C—OH, C—NH₂,C—NHR or C—NR₂;

R¹, R², R^(2′), R³, R^(3′), R⁴ are each independently H, halogen, alkyl,O-alkyl, OH, optionally protected O, methyl, H or F; and

R⁵ is an optionally protected OH or NH₂. This process may furthercomprise reacting

wherein Y is a halogen; and

wherein W⁴ is H or a metal-containing compound capable of metal-mediatedcross coupling. The OH and NH groups may be optionally deprotected.

In another embodiment, a process to make

comprises cyclising a compound having the formula

wherein each of Z¹-Z⁴ and Z⁶ is independently Z;

each of W¹ and W² is independently W;

wherein Y is halogen; and

Nu is a nucleophile. Preferably, A is O, CH₂ or optionally protected N;

X is optionally protected N, O, or S;

Nu is an alcohol, an alkylthiol, or an alkylamine;

W¹ is C (if p bond) or N (if no p bond);

W² is C, CH or N;

Z¹ and Z² are each independently CH, C-halogen, C-alkyl, C-aryl,C—O-alkyl, or C—S-alkyl;

Z³ is CH, C-alkyl, C-halogen, N, CNHR, CNH₂, CNR₂, C═O, or C═S;

Z⁴ is CH or C-halogen, C-alkyl, C-aryl, C—O-alkyl, C—S-alkyl, C—OH,C—NH₂, C—NHR or C—NR₂;

R¹, R², R^(2′), R³, R^(3′), R⁴ are each independently H, halogen, alkyl,O-alkyl, OH, optionally protected O, methyl, or F;

R⁵ is an optionally protected OH or NH₂; and

Z⁶, is CH₂, O, NH, NR or S. This process further comprises reacting

wherein W⁴ is H or a metal-containing compound capable of crosscoupling.

In addition, the compound of the invention may be further modified, forexample, to add various functional groups. In one embodiment, a compoundhaving the formula

may be modified using a nucleophile and/or electrophile to form acompound selected from the group consisting of:

wherein each of Z¹-Z⁴ are independently Z;

wherein each of W¹ and W² are independently W; and

wherein Nu is a nucleophile. Preferably, in this process

A is O, CH₂ or optionally protected N;

Nu is an alcohol, an alkylthiol, or an alkylamine;

W^(l) is C (if p bond) or N (if no p bond);

W² is C, CH or N;

Z¹ and Z² are each independently CH, C-halogen, C-alkyl, C-aryl,C—O-alkyl, or C—S-alkyl;

Z³ is CH, C-alkyl, C-halogen, N, CNHR, CNH₂, CNR₂, C═O, or C—S;

Z⁴ is CH or C-halogen, C-alkyl, C-aryl, C—O-alkyl, C—S-alkyl, C—OH,C—NH₂, C—NHR or C—NR₂;

R¹, R², R^(2′), R³, R^(3′), R⁴ are each independently H, halogen, alkyl,O-alkyl, OH, optionally protected O, methyl, or F;

R⁵ is an optionally protected OH or NH₂;

Q is O, NR, NH or S; and

R⁶ is alkyl, aryl, alkenyl or alkynyl.

In another embodiment, a pharmaceutical composition is providedcomprising a therapeutically effective amount of a compound of theformula (I) or a pharmaceutically acceptable salt or a prodrug thereof.

DETAILED DESCRIPTION OF THE INVENTION

The tricyclic nucleosides of the invention also include derivatives suchas nucleotide mimics and/or prodrugs thereof.

For example, in some embodiments, nucleotide mimics of the compounds ofthe Invention of formula (I) discussed above include:

a compound in which R⁵ is a monophosphate mimic of having formula (X) or(XI):

where X^(1′), X^(4′), and X^(6′) independently are O, S, NH, or NR; X²,X³, and X^(5′) are selected independently from the group consisting ofH, F, OH, SH, NH₂, NHOH, N₃, CN, ⁻BH₂GM⁺, ⁻BH₃M⁺, R, OR, SR, NHR, andNR₂. The substituents ⁻BH₂GM⁺ and BH₃M⁺ are ion pairs, which are linkedto phosphorus through the negatively charged boron. M⁺ is a cation.

In some embodiments, nucleotide mimics of the compounds of the Inventionof formula (I) discussed above include di- and tri-phosphate mimicsincluding:

a compound in which R⁵ is a di- or tri-phosphate moiety of formula(XII):

X², X³, and X⁴ are selected independently from the group consisting ofO, S, Se, NH and NR;

X⁵ and X are selected independently from the group consisting of O, S,Se, O₂, CY₂CO, CHOH, C(OH)₂, CH₂O, CH₂CH₂, CH₂CHNH₂, CH₂CH₂CHNH₂,CY₂OCY₂, CY₂, CRY, CY₂CY₂, CHR, CC, HC═CH, NH, NR, NOH, NOR, NNH₂, andNNHR;

Y is selected from the group consisting of H, F, Cl, Br, alkyl, alkenyl,and alkynyl, wherein alkyl, alkenyl, and alkynyl may each optionallycontain one or more heteroatoms;

R is selected from the group consisting of alkyl, alkenyl, alkynyl,aryl, acyl, and aralkyl, each optionally containing one or moreheteroatoms;

X⁷, X⁸, X⁹, and X¹⁰ are selected independently from the group consistingof H, F, OH, SH, NH₂, NHOH, NHOR, NHNH₂, NHNHR, CN, N₃, ⁻BH₃M⁺, ⁻BH₂GM⁺,R, OR, SR, SeH, SeR, NHR, and NR₂.

wherein n is 0 or 1. The substituents BH₂GM⁺ and ⁻BH₃M⁺are ion pairs,which are is linked to phosphorus through the negatively charged boron.M⁺ is a cation.

Additional nucleotide phosphate mimics and methods of making thephosphate mimics appropriate for the compounds of the invention aredescribed in PCT Publications WO 2003/072757 and WO 2003/073989, filedFeb. 28, 2003, which are incorporated herein by reference in theirentirety. Many nucleotide mimics of the present invention can beprepared by similar approaches as published or by using well-knownknowledge of organophosphorous chemistry. Generally, phosphate mimics ofthe nucleosides and nucleotides of the invention can inhibit enzymefunction without phosphorylation and/or have enhanced nuclease stabilityrelative to nucleotides with unmodified phosphate.

The term phosphate mimic, unless otherwise specified, refers to aphosphate analog, including, but not limited to, phosphonate,phosphothioate, phosphoselenate, selenophosphate, thiophosphate,P-boranophosphate, phosphoramidate, sulfamate, sulfonate, andsulfonamide and/or a combination thereof. Preferred embodiments of thephosphate mimics include phosphonate, phosphorothioate,methylphosphonate, fluromethylphosphonate, difluoromethylphosphonate,vinylphosphonate, phenylphosphonate, sulfonate, fluorophosphate,dithiophosphorothioate, 5′-methylenephosphonate,5′-difluoromethylenephosphonate, 5′-deoxyphosponate,5′-aminophosphoramidate, and 5′-thiophosphate. More preferred isphosphonate.

The terms diphosphate mimic and triphosphate mimic specifically refer toa diphosphate analog and a triphosphate analog, respectively, whichcomprises at least one of the phosphate mimics, one of the modificationsat the bridging site of diphosphate and triphosphate, or replacements ofnon-bridging phosphate oxygens. The modification at the bridging site,i.e., in the X⁵ and X⁶ positions of formula (XII), includes thereplacement of 0 by other atoms or functions such as S, Se, O₂, NH, NHR,NR, CH₂, CHF, CHCl, CHBr, CF₂, CCl₂, CBr₂, CHR, CYCO₂, CH₂O, CHOH,C(OH)₂, CH₂CH₂, CC, CH═CH, CH₂CH₂CHNH₂, CH₂CHNH₂, CY₂OCY₂, CY₂, CY₂CY₂,and CR₂ where R is selected from the group consisting of alkyl, alkenyl,alkynyl, aryl, acyl, and aralkyl each optionally containing one or moreheteroatoms. Non-bridging phosphate oxygens, e.g., in X⁷-X¹⁰ positionsof formula (XII) can be replaced by a variety of substituents includingH, F, OH, SH, NH₂, NHOH, NHOR, NHNH₂, NHNHR, CN, N₃, ⁻BH₃M⁺, R, OR, SR,SeH, SeR, NHR, NR₂, and R* where R is as defined herein, and wherein R*is a prodrug substituent. M⁺ is a cation preferably a pharmaceuticallyacceptable cation such as Ca²⁺, ammonium, trialkylammonium ortertaalkylammonium, e.g., NH₄ ⁺, Et₃NH⁺, Bu₃NH⁺, and Bu₄N⁺.

The α-P, β-P, and γ-P in the diphosphate mimics and triphosphate mimicsmay independently adopt either R or S configurations when they become achiral phosphorus.

In some embodiments, the tricyclic nucleosides and nucleotides ofinvention also include their prodrug derivatives. In addition to thosedescribed herein, prodrug derivatives of nucleosides, nucleotides andnucleotide phosphate mimics and methods of making the prodrugsappropriate for use in the present invention are described in PCTPublications WO 2003/072757 and WO 2003/073989. Such prodrugmodification is to enhance drug absorption and/or drug delivery intocells.

In one embodiment, such compounds of the invention include prodrugs(e.g., one or more of an —OH group of a mono, di or triphosphate, one ormore of X^(2′), X^(3′) or X^(5′), or X⁷-X¹⁰ in (XII) is a prodrugsubstituent R*) of the compounds of formula (I) discussed herein.

R* is a prodrug substituent which may be conjugated to one or moreX⁷-X¹⁰ positions. The term prodrug, unless otherwise specified, refersto a masked (protected) form of a nucleotide, such as a mimic of formula(X) or (XI) that is formed when one or more of X^(2′), X^(3′) or X^(5′)is R* or to a masked (protected) form of a nucleotide mimic of formula(XII) when one or more of X⁷-X¹⁰ is R*. The prodrug of a nucleoside5′-monophosphate mimic can mask the negative charges of the phosphatemimic moiety entirely or partially, mask the negative charges of thedi-phosphate (X⁷, X⁸, X¹⁰) mimic or tri-phosphate (X⁷-X¹⁰) mimic moietyor phosphate moiety, entirely or partially, or mask a heteroatomsubstituted alkyl, aryl or aryalkyl (W′, see below) attached to aphosphate or phosphate mimic moiety in order to enhance drug absorptionand/or drug delivery into cells. The prodrug can be activated either bycellular enzymes such as lipases, esterases, reductases, oxidases,nucleases or by chemical cleavage such as hydrolysis to release(liberate) the nucleotide mimic after the prodrug enters cells. Prodrugsare often referred to as cleavable prodrugs. Prodrugs substituentsinclude, but are not limited to: proteins; antibiotics (and antibioticfragments); D- and L-amino acids attached to a phosphate moiety or aphosphate mimic moiety via a carbon atom (phosphonates), a nitrogen atomphosphoamidates), or an oxygen atom (phosphoesters); peptides (up to 10amino acids) attached to a phosphate moiety or a phosphate mimic moietyvia a carbon atom (phosphonates), a nitrogen atom (phosphoamidates), oran oxygen atom (phosphoesters); drag moieties attached to a phosphatemoiety or a phosphate mimic moiety via a carbon atom (phosphonates), anitrogen atom (phosphoamidates), or an oxygen atom (phosphoesters);steroids; cholesterols; folic acids; vitamins; polyamines;carbohydrates; polyethylene glycols (PEGs); cyclosaligenyls; substituted4 to 8-membered rings, with or without heteroatom substitutions,1,3-phosphoamidate attachments to a terminal phosphate or phosphatemimic moiety (γ or β) or connecting between an α, β or β,γ phosphatemoiety or phosphate mimic moiety; acylthioethoxy, (SATE) RCOSCH₂CH₂O—;RCOSCH₂CH₂O—W′—O—; RCOSCH₂CH₂O—W′—S—; RCOSCH₂CH₂O—W′—NH—;RCOSCH₂CH₂O—W′—; RCOSCH₂CH₂O—W′—CY₂—; acyloxymethoxy, RCOOCH₂O—;RCOOCH₂O—W′—O—; RCOOCH₂O—W′—S—; RCOOCH₂O—W′—NH—; RCOOCH₂O—W′—;RCOOCH₂O—W′—CY₂—; alkoxycarbonyloxymethoxy, ROCOOCH₂O—; ROCOOCH₂O—W′—O—;ROCOOCH₂O—W′—S—; ROCOOCH₂O—W′—NH—; ROCOOCH₂O—W′—; ROCOOCH₂O—W′—CY₂—;acylthioethyldithioethoxy (DTE) RCOSCH₂CH₂SSCH₂CH₂O—;RCOSCH₂CH₂SSCH₂CH₂—W′—; RCOSCH₂CH₂SSCH₂CH₂O—W′—O—;RCOSCH₂CH₂SSCH₂CH₂O—W′—S—; RCOSCH₂CH₂SSCH₂CH₂O—W′—NH—;RCOSCH₂CH₂SSCH₂CH₂O—CY₂—; acyloxymethylphenylmethoxy (PAOB)RCO₂—C₆H₄—CH₂—O—; RCO₂—C₆H₄—CH₂—O—W′—; RCO₂—C₆H₄—CH₂—O—W′—O—;RCO₂—C₆H₄—CH₂—O—W′—S—; RCO₂—C₆H₄—CH₂—O—W′—NH—; RCO₂—C₆H₄—CH₂—O—W′—CY₂—;1,2-O-diacyl-glyceryloxy, RCOO—CH₂—CH(OCOR)—CH₂O—;1,2-O-dialkyl-glyceryloxy, RO—CH₂—CH(OR)—CH₂O—;1,2-S-dialkyl-glyceryloxy, RS—CH₂—CH(SR)—CH₂O—;1-O-alkyl-2-O-acyl-glyceryloxy, RO—CH₂—CH(OCOR)—CH₂O—;1-S-alkyl-2-O-acyl-glyceryloxy, RS—CH₂—CH(OCOR)—CH₂O—;1-O-acyl-2-O-alky-glyceryloxy, RCOO—CH₂—CH(OR)—CH₂O—;1-O-acyl-2-S-alky-kglyceryloxy, RCOO—CH₂—CH(SR)—CH₂O—; any substituentattached via a carbon, nitrogen or oxygen atom to a nucleoside di- ortri-phosphate mimic that liberates the di- or tri-phosphate mimic invivo.

A combination of prodrug substituents may be attached (conjugated) toone or more X^(2′), X^(3′) and X^(5′) positions on a nucleosidemono-phosphate mimic or to one or more X⁷-X¹⁰ positions on a nucleosidedi- or tri-phosphate mimic. W′is alkyl, aryl, aralkyl as described aboveor a heterocycle. Preferred prodrug substituents (R*) in positionsX^(2′), X^(3′) or X^(5′) include 2,3-O-diacylglyceryloxy,2,3-O-dialkylglyceryloxy, 1-O-alkyl-2-O-acylglyceryloxy,1-O-acyl-2-O-alkylglyceryloxy, 1-S-alkyl-2-O-acyl-1-thioglyceryloxy,acyloxymethoxy, S-acyl-2-thioethoxy, S-pivaloyl-2-thioethoxy,acyloxymethoxy, pivaloyloxymethoxy, alkoxycarbonyloxymethoxy,S-alkyldithio-S′-ethyoxy acyloxymethoxy, S-acyl-2-thioethoxy,S-pivaloyl-2-thioethoxy, pivaloyloxymethoxy, alkoxycarbolnyloxymethoxy,and S-alkyldithio-S′-ethyoxy.

The term moiety, unless otherwise specified, refers to a portion of amolecule. Moiety may be, but is not limited to, a functional group, anacyclic chain, a prodrug masking group, an aromatic ring, acarbohydrate, a carbocyclic ring, or a heterocycle.

The term base, unless otherwise specified, refers to the base moiety ofa nucleoside or nucleotide. The base moiety is the heterocycle portionof a nucleoside or nucleotide. The base moiety of a nucleotide offormula (I) is a tricyclic heterocycle represented by formulae II-III.Preferably, the base moiety of a nucleotide of formula (I) may be atricyclic heterocycle represented by any one of formulae IV-IX. Thenucleoside base is attached to the sugar moiety of a nucleoside in suchways that both β-D- and β-L-nucleoside can be produced.

The term sugar refers to the ribofuranose portion of a nucleoside. Thesugar moiety of formula (I) nucleosides and nucleotides mimics and/orprodrugs thereof may contain one or more substituents at their C1-, C2-,C3- and C4-position of the ribofuranose. Substituents may direct toeither the α- or β-face of the ribofuranose. The nucleoside base thatcan be considered as a substituent at the C-1 position of theribofuranose directs to the β-face of the sugar. The β-face is the sideof a ribofuranose on which a purine or pyrimidine base of naturalβ-D-nucleosides is present. The α-face is the side of the sugar oppositeto the β-face. The sugar moiety of the present invention is not limitedto a ribofuranose and its derivatives, instead it may be a carbohydrate,a carbohydrate analog, a carbocyclic ring, or other ribofuranoseanalogs.

The term sugar-modified nucleoside or nucleotide refers to a nucleosideor nucleotide containing a modified sugar moiety.

The term base-modified nucleoside or nucleotide refers to a nucleosideor nucleotide containing a modified base moiety.

The term alkyl, unless otherwise specified, refers to a saturatedstraight, branched, or cyclic hydrocarbon of C1 to C18. Alkyls mayinclude, but are not limited to, methyl, ethyl, n-propyl, isopropyl,cyclopropyl, n-butyl, isobutyl, t-butyl, cyclobutyl, n-pentyl,isopentyl, neopentyl, cyclopentyl, n-hexyl, cyclohexyl, dodecyl,tetradecyl, hexadecyl, or octadecyl.

The term alkenyl, unless otherwise specified, refers to an unsaturatedhydrocarbon of C2 to C18 that contains at least one carbon-carbon doublebond and may be straight, branched or cyclic. Alkenyls may include, butare not limited to, olefinic, propenyl, allyl, 1-butenyl, 3-butenyl,1-pentenyl, 4-pentenyl, 1-hexenyl, or cyclohexenyl.

The term alkynyl, unless otherwise specified, refers to an unsaturatedhydrocarbon of C2 to C18 that contains at least one carbon-carbon triplebond and may be straight, branched or cyclic. Alkynyls may include, butare not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, or3-butynyl.

The term aryl, unless otherwise specified, refers to an aromatic moietywith or without one or more heteroatoms. Aryls may include, but are notlimited to, phenyl, biphenyl, naphthyl, pyridinyl, pyrrolyl, andimidazolyl optionally containing one or more substituents. Thesubstituents may include, but are not limited to, hydroxy, amino, thio,halogen, cyano, nitro, alkoxy, alkylamino, alkylthio, hydroxycarbonyl,alkoxycarbonyl, or carbamoyl.

The term aralkyl, unless otherwise specified, refers to a moiety thatcontains both an aryl and an alkyl, an alkenyl, or an alkynyl. Aralkylscan be attached through either the aromatic portion or the non-aromaticportion. Aralkyls may include, but are not limited to, benzyl,phenethyl, phenylpropyl, methylphenyl, ethylphenyl, propylphenyl,butylphenyl, phenylethenyl, phenylpropenyl, phenylethynyl, orphenylpropynyl.

The term acyl, unless otherwise specified, refers to alkylcarbonyl.Acyls may include, but are not limited to, formyl, acetyl, fluoroacetyl,difluoroacetyl, trifluoroacetyl, chloroacetyl, dichloroacetyl,trichloroacetyl, propionyl, benzoyl, toluoyl, butyryl, isobutyryl, orpivaloyl.

The term heteroatom refers to oxygen, sulfur, nitrogen, or halogen. Whenone or more heteroatoms are attached to alkyl, alkeneyl, alkynyl, acyl,aryl, or arakyl, a new functional group may be produced. For instance,when one or more heteroatoms are attached to an alkyl, substitutedalkyls may be produced, including, but not limited to, fluoroalkyl,chloroalkyl, bromoalkyl, iodoalkyl, alkoxy, hydroxyalkyl, alkylamino,aminoalkyl, alkylthio, thioalkyl, azidoalkyl, cyanoalkyl, nitroalkyl,carbamoylalkyl, carboxylalkyl, and acylalkyl.

Benzoazulenes, such as benzo[cd]azulene refer to a class of tricycliccompounds having a fused, 5, 6, and 7-membered rings that may containone or more heteroatoms, preferably O or N, in the backbone of the ring,and thus derivatives of benzoazulene is also included in such ten-n.

The term halogen or halo refers to fluorine, chlorine, bromine, oriodine.

The term function refers to a substituent. Functions may include, butare not limited to, hydroxy, amino, sulfhydryl, azido, cyano, halo,nitro, hydroxyamino, hydroxycarbonyl, alkoxycarbonyl, or carboxyl eitherprotected or unprotected.

R may be formula (I) is a univalent substituent and present on the base,sugar and other moieties. R may be selected from the group consisting ofalkyl, alkenyl, alkynyl, aryl, acyl, and aralkyl optionally containingone or more heteroatoms, which are as defined above.

A “protecting group” for example for O, S, or N such as hydroxy or NH₂,includes acyl groups, silyl groups, and the like. Suitable protectinggroups are described by Greene, T. W., et al., in Protecting Groups inOrganic Synthesis. 2^(nd) Ed., John Wiley & Sons, Inc. (1991),incorporated herein by reference. “Nuceophile” and “electrophile” havetheir ordinary meaning in the art. Examples of preferred nucleophilesare alcohols, alkylthiols or alkylamines, which may be optionallyprotected. “Nu” refers to both the free nucleophiles and thenucleophiles as attached to the tricyclic compound of the invention.Thus, a nucleophile may be, for example, DMF or MeO-. Preferably, anucleophile may be optionally protected N, O or S.

In addition to using prodrug approaches, the delivery of the nucleosidesand nucleotides may be assisted by using a therapeutically acceptablecarrier such as liposomal suspensions, cationic lipids, and polyimines.In compounds of formula (I) where a chiral center is present, theinvention encompasses enantiomers, or stereoisomers and mixturesthereof, such as enantiomerically enriched mixtures.

The term “infection” or “microbial infection” refers to the infectioncaused by an infectious agent or microbe, such as bacteria, parasite(including protozoan), virus or fungus (including unicellular andmulticellular). Examples of microbes that cause such infection include:Acanthamoeba, African Sleeping Sickness (Trypanosomiasis), amebiasis,American Trypanosomiasis (Chagas Disease), Bilharzia (Schistosomiasis),cryptosporidiosis (diarrheal disease, Cryptosporidium Parvum),Giardiasis (diarrheal disease, Giardia laniblia), hepatitis A, B, C, D,E, leishmaniasis (skin sores and visceral), malaria (Plasmodiumfalciparum), Salmonella enteritides infection (stomach cramps, diarrheaand fever), tuberculosis (mycobacterium tuberculosis), varicella(chicken pox), yellow fever, pneumonias, urinary tract infections(Chlamydia and Mycoplasma), meningitis and meningococcal septicemia,skin and soft tissue infections (Staphylococcus aureus), lowerrespiratory tract infections (bacterial pathogens or hepatitis C).

Common infections caused by microbes are further outlined in thefollowing chart: Infection Bacteria Fungus Protozoa Virus AIDS XAthlete's Foot X Chicken Pox X Common Cold X Diarrheal Disease X X X FluX Genital Herpes X Malaria X X Meningitis X Pneumonia X X Sinusitis X XSkin Disease X X X X Strep Throat X Tuberculosis X Urinary TractInfections X Vaginal Infections X X Viral Hepatitis XChemical Synthesis

The novel nucleosides and nucleotides, and prodrugs thereof, of thepresent invention can be prepared by those who are skillful in syntheticorganic and nucleoside chemistry using established synthetic methodology(Chemistry of Nucleosides and Nucleotides Vol. 1, 2, 3, edited byTownsend, Plenum Press; Handbook of Nucleoside Synthesis by VorbrüggenRuh-Pohlenz, John Wiley & Sons, Inc., 2001; The Organic Chemistry ofNucleic Acids by Yoshihisa Mizuno, Elsevier, 1986). The nucleosides ofthe present invention can be converted to their correspondingmonophosphate, diphosphate, and triphosphate by establishedphosphorylation procedures. Similarly, known methods in the art can beused to synthesize the nucleotide prodrugs and phosphate mimics. Thefollowing schemes and descriptions serve as representative syntheses ofthe nucleosides of the present invention. As such, other compounds suchas those having -L-R^(4′) groups other than CH₂R⁵ may similarly be made.

Gycosyl benzo[cd]azulenes can be prepared by modification of optionallyprotected and functionalized 7-deazapunrine analogues followed byStille, Heck or other metal-mediated cross coupling chemistry tointroduce an α,β-unsaturated ester or other carbonyl group. Such processallows for stereoselective synthesis of an intermediate capable ofefficient cyclisation to the inventive compound. Any compound capable ofmetal-mediated cross coupling may be used, such as a tin derivative,such as trialkyltin. More preferably tributyltin. Cyclisation andoptional deprotection of the product delivers the target nucleosidewhich contains the benzo[cd]azulene, a key element of the invention.

In Scheme 1, preferably A is O, CH₂ or optionally protected N; Y ishalogen; W¹ is C (if p bond) or N (if no p bond); W² is C, CH or N; W⁴is H or trialkyltin; Z¹ and Z² are each independently CH, C-halogen,C-alkyl, C-aryl, C—O-alkyl or C—S-alkyl; Z³ is CH, C-alkyl, C-halogen,N, CNHR, CNH₂, CNR₂, C═O, or C═S; Z⁴ is CH, C-halogen, C-alkyl, C-aryl,C—O-alkyl, C—S-alkyl, C—OH, C—NH₂, C—NHR or C—NR₂; R¹, R², R^(2′), R³,R^(3′), R⁴ are each independently H, halogen, alkyl, O-alkyl, OH,optionally protected O, methyl, H or F; R⁵ is an optionally protected OHor NH₂.

An alternative to the use of vinyl esters in the metal-mediated crosscoupling is the use of vinyl nitriles. In this case, cyclisation andoptional deprotection of the product delivers the target nucleoside inthe form of an amidine-containing benzo[cd]azulene.

In Scheme 2, preferably A is O, CH₂ or optionally protected N; Y ishalogen; W¹ is C (if p bond) or N (if no p bond); W² is C, CH or N; W⁴is H or trialkyltin; Z¹ and Z² are each independently CH, C-halogen,C-alkyl, C-aryl, C—O-alkyl or C—S-alkyl; Z³ is CH, C-alkyl, C-halogen,N, CNHR, CNH₂, CNR₂, C═O, or C═S; Z⁴ is CH, C-halogen, C-alkyl, C-aryl,C—O-alkyl, C—S-alkyl, C—OH, C—NH₂, C—NHR or C—NR₂; R¹, R², R^(2′), R³,R^(3′), R⁴ are each independently H, halogen, alkyl, O-alkyl, OH,optionally protected O, methyl, or F; R⁵ is an optionally protected OHor NH₂.

Similar cross-coupling methodology can be applied where the alkenepartner in the cross-coupling reaction is equipped with a nucleophilewhich may be present in protected form. Intramolecular nucleophilicsubstitution by the pendant nucleophile delivers the requisitebenzo[cd]azulene. SNAr displacement reactions are an example of suchnucleophilic substitutions.

In Scheme 3, preferably A is O, CH₂ or optionally protected N; Y ishalogen; X is optionally protected N, O or S; W¹ is C (if p bond) or N(if no p bond); W² is C, CH or N; W⁴ is H or trialkyltin; Z¹ and Z² areeach independently CH, C-halogen, C-alkyl, C-aryl, C—O-alkyl orC—S-alkyl; Z³ is CH, C-alkyl, C-halogen, N, CNHR, CNH₂, CNR₂, C═O, orC═S; Z⁴ is CH or C-halogen, C-alkyl, C-aryl, C—O-alkyl, C—S-alkyl,C—NH₂, C—NHR or C—NR₂; Z⁶¹S CH₂, O, NH or NR; R¹, R², R^(2′), R³,R^(3′), R⁴ are each independently H, halogen, alkyl, O-alkyl, OH,optionally protected O, methyl, or F; R⁵ is an optionally protected OHor NH₂; Nu is optionally protected N, O, or S.

An alternative approach to the use of metal-mediated cross couplingsinvolves halogen-metal exchange of a suitably functionalised7-deazapurine derivative and interception of the so-formedorganometallic intermediate with a suitable electrophile. If thiselectrophile is also equipped with an optionally protected nucleophile,intramolecular nucleophilic substitution by the pendant nucleophiledelivers the requisite benzo[cd]azulene.

In Scheme 4, preferably A is O, CH₂ or optionally protected N; Y ishalogen; X is optionally protected N, O or S; W¹ is C (if p bond) or N(if no p bond); W² is C, CH or N; W⁴ is H or trialkyltin; Z³ is CH,C-alkyl, C-halogen, N, CNHR, CNH₂, CNR₂, C═O, or C═S; Z⁴ is CH orC-halogen, C-alkyl, C-aryl, C—O-alkyl, C—S-alkyl, C—NH₂, C—NHR or C—NR₂;Z⁵ Z⁶ Z⁹ are each independently CH₂, O, NH or NR; R¹, R², R^(2′), R³,R^(3′), R⁴ are each independently H, halogen, alkyl, O-alkyl, OH,optionally protected O, methyl, or F; R⁵ is an optionally protected OHor NH₂; Nu is a nucleophile such as optionally protected N, O, or S; Lvis Leaving group.

Gycosyl benzo[cd]azulenes can alternatively be prepared by glycosylationof intact benzo[cd]azulenes as shown in Scheme 2. Conditions used forsuch glycosidations are well known to practitioners of the art and canbe found in the references cited above.

In Scheme 5, preferably A is O, CH₂ or optionally protected N; Z¹ and Z²are each independently CH, C-halogen, C-alkyl, C-aryl, C—O-alkyl orC—S-alkyl; Z³ is CH, C-alkyl, C-halogen; N, CNHR, CNH₂, CNR₂, C═O, orC═S; Z⁴ is CH, C-halogen, C-alkyl, C-aryl, C—O-alkyl, C—S-alkyl, C—OH,C—NH₂, C—NHR or C—NR₂; R², R^(2′), R³, R^(3′), R⁴ are each independentlyH, halogen, alkyl, O-alkyl, OH, optionally protected O, methyl, or F; R⁵is an optionally protected OH or NH₂; Lv is Leaving group.

Certain gycosyl benzo[cd]azulene-7-ones can be modified by well knownfunctional group interconversions (FGIs). For example, the 8,9-doublebond can be manipulated by hydrogenation, addition oraddition-elimination processes. The 7-carbonyl function can also beconverted into a 7-thiocarbonyl or undergo O-alkylation.

When equipped with an appropriate leaving group such as a chlorine atomthat the 4-position of the gycosyl benzo[cd]azulene framework, a rangeof nucleophiles can engage in nucleophilic substitutions at thisposition. Suitable nucleophiles include alcohols, alkyl thiols andalkylamines.

In Scheme 6, preferably A is O, CH₂ or optionally protected N; Nu isnucleophile such as optionally protected N, O, or S; W¹ is C (if p bond)or N (if no p bond); W² is C, CH or N; Z¹ and Z² are each independentlyCH, C-halogen, C-alkyl, C-aryl, C—O-alkyl, or C—S-alkyl; Z³ is CH,C-alkyl, C-halogen, N, CNHR, CNH₂, CNR₂, C═O, or C═S; Z⁴ is CH orC-halogen, C-alkyl, C-aryl, C—O-alkyl, C—S-alkyl, C—OH, C—NH₂, C—NHR orC—NR₂; R¹, R², R^(2′), R³, R^(3′), R⁴ are each independently H, halogen,alkyl, O-alkyl, OH, optionally protected O, methyl, or F; R⁵ is anoptionally protected OH or NH₂; X is O or S; R⁶ is alkyl, aryl, alkenylor alkynyl.

When equipped with a 4-amino group the gycosyl benzo[cd]azuleneframework can be modified by interception of a derived diazonium ionusing standard techniques.

In Scheme 7, preferably A is O, CH₂ or optionally protected N; W¹ is C(if π bond) or N (if no π bond); W² is C, CH, or N; Z¹ and Z² are eachindependently CH, C-halogen, C-alkyl, C-aryl, C—O-alkyl or C—S-alkyl; Z³is CH, C-alkyl, C-halogen, N, CNHR, CNH₂, CNR₂, C═O, or C═S; Y ishalogen; R¹, R², R^(2′), R³, R^(3′), R⁴ are each independently H,halogen, alkyl, O-alkyl, OH, optionally protected O, methyl, or F; R⁵ isOH, NH₂ or optional protecting group; R⁶, R⁷, and R⁸ are eachindependently alkyl, aryl, alkenyl or alkynyl.

In Scheme 8, preferably A is O, CH₂ or optionally protected N; W¹ is C(if π bond) or N (if no π bond); W² is C, CH or N; Z¹ and Z² are eachindependently CH, C-halogen, C-alkyl, C-aryl, C—O-alkyl or C—S-alkyl; Z³is CH, C-allyl, C-halogen, N, CNHR, CNH₂, CNR₂, C═O, or C═S; Y ishalogen; R¹, R², R^(2′), R³, R^(3′), R⁴ are each independently H,halogen, alkyl, O-alkyl, OH, optionally protected O, methyl, or F; R⁵ isoptionally protected OH or NH₂.

The compounds described here can be converted into their correspondingmono-, di- and triphosphates using well established methods.Furthermore, prodrugs of mono-, di- and triphosphates can be prepared inorder to optimise the biological efficacy of these phosphorylatedcompounds. Methods for preparing such prodrugs are well known in the art(see Wagner, C. R., et al. Med. Res. Rev., 2000, 20, 417-451).

In Scheme 9, preferably A is O, CH₂ or optionally protected N; R¹, R²,R^(2′), R³, R^(3′), R⁴ are each independently H, halogen, alkyl,O-alkyl, OH, optionally protected O, methyl or F, Base is as describedherein.

An alternative to the use of phosphates and prodrugs of these is the useof phosphate mimics and their prodrugs (for prodrugs, see Wagner, C. R.,et al. Med. Res. Rev., 2000, 20, 417-451). One such phosphate mimic isshown below and this can be prepared using appropriately protectednucleosides and known conditions.

Methods of preparing tri-, di, and mono-phosphate mimics useful formaking compounds of the invention is found in WO 2003/072757 and WO2003/073989 filed Feb. 28, 2003. A representative scheme is describedabove.

In Scheme 10, preferably A is O, CH₂ or optionally protected N; R¹, R²,R^(2′), R³, R^(3′), R⁴ are each independently H, halogen, alkyl,O-alkyl, OH, optionally protected O, methyl or F; X′ is O, S, NH, CF₂,CHF, CClH, CBr₂ or CHBr; Base is as described herein.

Biological Assays

Antiviral assays are conducted according to published, widely usedprotocols. In order to obtain the therapeutic index, compound-inducedcytotoxicity to host cells is also measured in parallel with antiviralactivities. To determine the mode of action of antiviral nucleosides thecorresponding nucleoside triphosphates are subject to enzyme-basedassays for the inhibition of viral polymerases according to knownprotocols (Ranjith-Kumar et al. J. Virol. 2001, 75, 8615; Dhanak et al.J. Biol. Chem. 2002, 277, 38322-38327). Some compounds of the presentinvention showed K_(i) values of less than 1 μM against HCV NS5B.

Since the replicon RNA replication mimics the replication of HCV RNA ininfected hepatocytes, compounds that have the inhibitory effects inreplicon assays are potentially useful as anti-HCV drugs. The HCVreplicon-containing cell lines (Randall and Rice, Current Opinion inInfectious Diseases 2001, 14, 743) are used for the identification ofpotential anti-HCV compounds. Among them is a widely used subgenomicreplicon system developed by Lohmann et al. (Science 1999, 285, 110; J.General Virol. 2000, 81, 1631; J. Virol. 2001, 75, 1437, 2002, 76,4008). Some compounds of the present invention showed potent anti-HCVactivity with EC₅₀ values of low μM.

Widely used protocols developed by Korba et al. (Antiviral Res. 1992,19, 55), and Pai et al. (Antimicrobial Agents Chemother. 1996, 40, 380)are useful for the determination of in vitro anti-HBV activity.

Anti-HIV assays can be conducted according to the protocols developed bySchinazi et al. (Antimicrobial Agents Chemother. 1990, 34, 1061; 1992,36, 2423; 1993, 37, 875) or other widely used protocols (Kimpton et al.J. Virol. 1992, 66, 2232; Chan et al. J. Med. Chem. 2001, 44, 1866).

Biological Applications and Administration

The nucleosides, nucleotide mimics and/or their prodrugs of the presentinvention may be useful for the inhibition of a variety of enzymesincluding, but not limited to, DNA or RNA polymerases, helicases,ribonucleotide reductases, protein kinases, and telomerases and for themodulation of G-proteins, P2 purinergic receptors and the allostericsites of a variety of enzymes.

The nucleosides, nucleotide mimics and/or their prodrugs of the presentinvention are useful as human therapeutics for the treatment ofinfectious diseases caused by viruses including, but not limited to,HIV, HBV, HCV, HDV, HSV, HCMV, small pox, West Nile virus, SARS virus,influenza viruses, measles, rhinovirus, RSV, VZV, EBV, vaccinia virus,and papilloma virus.

The nucleosides, nucleotide mimics and/or their prodrugs of the presentinvention are useful for the treatment of infectious diseases caused byinfectious agents such as parasites, bacteria and fungi.

Those nucleosides, nucleotide mimics and/or their prodrugs that havepotent cytotoxicities to fast-dividing cancerous cells are useful forthe treatment of proliferative disorders, including, but not limited to,lung cancer, liver cancer, prostate cancer, colon cancer, breast cancer,ovarian cancer, melanoma, and leukemia.

As the ligands of P2 receptors and G-proteins as well as the inhibitorsof protein kinases, the nucleosides, nucleotide mimics and/or theirprodrugs of the present invention are useful for the treatment of a widerange of other diseases and disorders such as inflammatory diseases,autoimmune diseases, Type 2 diabetes, and cardiovascular diseases.

In order to overcome drug resistance, combination therapies are widelyused in the treatment of infectious diseases and proliferativedisorders. The nucleosides, nucleotide mimics and/or their prodrugs ofthe present invention may be therapeutically administered as a singledrug, or alternatively may be administered in combination with one ormore other active chemical entities to form a combination therapy. Theother active chemical entities may be a small molecule, a polypeptide,or a polynucleotide.

The pharmaceutical composition of the present invention comprises atleast one of the compounds represented by the formulas herein orpharmaceutically acceptable salts, esters or prodrugs thereof as activeingredients. The compositions include those suitable for oral, topical,intravenous, subcutaneous, nasal, ocular, pulmonary, and rectaladministration. The compounds of the invention can be administered tomammalian individuals, including humans, as therapeutic agents.

Accordingly, the compounds of the invention are useful as anti-microbialinfection agents. The present invention provides a method for thetreatment of a patient afflicted with an infection comprisingadministering to the patient a therapeutically effective anti-microbialamount of a compound of the invention. The term “microbe infection” asused herein refers to an abnormal state or condition characterized bymicrobial transformation of cells, microbial replication and/ormicrobial proliferation. Microbial infections for which treatment with acompound of the invention will be particularly useful include themicrobes mentioned above.

The term “treat” as in “to treat a disease” is intended to include anymeans of treating a disease in a mammal, including (1) preventing thedisease, i.e., avoiding any clinical symptoms of the disease, (2)inhibiting the disease, that is, arresting the development orprogression of clinical symptoms, and/or (3) relieving the disease,i.e., causing regression of clinical symptoms.

For example, the compounds of the invention are useful as antiviralagents. The present invention provides a method for the treatment of apatient afflicted with a viral infection comprising administering to thepatient a therapeutically effective antiviral amount of a compound ofthe invention. The term “viral infection” as used herein refers to anabnormal state or condition characterized by viral transformation ofcells, viral replication and proliferation. Viral infections for whichtreatment with a compound of the invention will be particularly usefulinclude the viruses mentioned above.

A “therapeutically effective amount” of a compound of the inventionrefers to an amount which is effective, upon single or multiple doseadministration to the patient, in controlling the growth of e.g., themicrobe or tumor or in prolonging the survivability of the patientbeyond that expected in the absence of such treatment. As used herein,“controlling the growth” refers to slowing, interrupting, arresting orstopping the microbial or proliferative transformation of cells or thereplication and proliferation of the microbe and does not necessarilyindicate a total elimination of e.g., the microbe or tumor.

Accordingly, the present invention includes pharmaceutical compositionscomprising, as an active ingredient, at least one of the compounds ofthe invention in association with a pharmaceutical carrier. Thecompounds of this invention can be administered by oral, parenteral(intramuscular, intraperitoneal, intravenous (IV) or subcutaneousinjection), topical, transdermal (either passively or usingiontophoresis or electroporation), transmucosal (e.g., nasal, vaginal,rectal, or sublingual) or pulmonary (e.g., via dry powder inhalation)routes of administration or using bioerodible inserts and can beformulated in dosage forms appropriate for each route of administration.

Solid dosage forms for bral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, or starch. Such dosage forms can alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., lubricating, agents such as magnesium stearate. In thecase of capsules, tablets, and pills, the dosage forms may also comprisebuffering agents. Tablets and pills can additionally be prepared withenteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, with the elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized by, forexample, filtration through a bacteria retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured using sterile water, or some other sterile injectablemedium, immediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as cocoa butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with standardexcipients well known in the art.

Topical formulations will generally comprise ointments, creams, lotions,gels or solutions. Ointments will contain a conventional ointment baseselected from the four recognized classes: oleaginous bases;emulsifiable bases; emulsion bases; and water-soluble bases. Lotions arepreparations to be applied to the skin or mucosal surface withoutfriction, and are typically liquid or semiliquid preparations in whichsolid particles, including the active agent, are present in a water oralcohol base. Lotions are usually suspensions of solids, and preferably,for the present purpose, comprise a liquid oily emulsion of theoil-in-water type. Creams, as known in the art, are viscous liquid orsemisolid emulsions, either oil-in-water or water-in-oil. Topicalformulations may also be in the form of a gel, i.e., a semisolid,suspension-type system, or in the form of a solution.

Finally, formulations of these drugs in dry powder form for delivery bya dry powder inhaler offer yet another means of administration. Thisovercomes many of the disadvantages of the oral and intravenous routes.

The dosage of active ingredient in the compositions of this inventionmay be varied; however, it is necessary that the amount of the activeingredient shall be such that a suitable dosage form is obtained. Theselected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment desired.Generally, dosage levels of between 0.001 to 10 mg/kg of body weightdaily are administered to mammals.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toprepare and use the compounds disclosed and claimed herein.

In order to overcome drug resistance, combination therapies are widelyused in the treatment of viral infections. The nucleoside analogs,corresponding 5′-monophosphate, 5′-diphosphate, 5′-triphosphate, andprodrugs thereof of the present invention may be therapeuticallyadministered as a single drug, or alternatively they may be administeredin combination with one or more other active chemical entities to form acombination therapy. The other active chemical entities may be a smallmolecule, a polypeptide, or a polynucleotide.

All references mentioned herein are incorporated herein by reference intheir entirety.

The following Examples are offered to illustrate but not to limit theinvention.

EXAMPLES Example 12-(2-C-methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-1,2,3,5,6-pentaazabenzo[cd]azulene-7-one(1.7)

For the preparation of tricyclic nucleoside 1.7,4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidine 1.2 was prepared by theiodination of 4-amino-1H-pyrazolo[3,4-d]pyrimidine 1.1 usingN-iodosuccinimide in DMF at 80° C. The glycosylation of 1.2 withcommercially available 2-C-methyl-tetrabenzoyl ribofuranose 13 inboiling nitromethane in the presence of boron trifluoride etherate gave2′C-methyl nucleoside 1.4 in 65% yield after purification. Removal ofester blocking groups with ammonia in MeOH provided the free nucleoside1.5 in 83% isolated yield. The use of methyl acrylate in the Pd[0]catalyzed cross coupling reaction of 1.5 afforded the 7-alkenylnucleoside 1.6. The treatment of compound 1.6 with NaOMe/MeOH resultedin an intramolecular cyclization, yielding the target tricyclicnucleoside 1.7.

Example 1, Step-A 4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidine (1.2)

4-Amino-1H-pyrazolo[3,4-d]pyrimidine was prepared according to thepublished method (J. Med. Chem. 1993, 36, 3424-3430).

Example 1, Step-B4-Amino-3-iodo-1-(2-C-methyl-2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine(1.4)

1-O-Acetyl-2-C-methyl-2,3,5-tri-O-benzoyl-D-ribofuranose (1.0 g, 1.72mmol) was dissolved in anhydrous nitromethane (10.0 mL) and to thissolution compound 1.2 (312 mg, 1.21 mmol) was added. The resultingsuspension was brought to reflux and borontrifluoride etherate (0.23 mL,1.78 mmol) was added. The suspension became a clear solution, which washeated at reflux for 2 hr. The mixture was cooled, the solvents wereevaporated and the off-white foamy residue was dissolved in ethylacetate and then poured with stirring into aq. sat. NaHCO₃. The aqueouslayer was extracted twice with ethyl acetate and the combined organiclayers were dried (Na₂SO₄), filtered and evaporated to give an off-whitesolid. This material was purified by flash column chromatography onsilica gel using CH₂Cl₂ to 2-3% MeOH in dichoromethane as eluent to givethe desired compound 1.4 (811 mg) as a yellow foam.

Example 1, Step-C4-Amino-3-iodo-1-(2-C-methyl-β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine(1.5)

A solution of compound 1.4 (540 mg, 0.75 mmol) in MeOHic NH₃ (120 mL,saturated at 0° C.) was stirred in a bomb at 45° C. for 16 hr. Themixture was evaporated to dryness and then the residue co-evaporatedwith additional MeOH. Purification by silica gel column chromatographyusing 6-14% MeOH in dicholoromethane as eluent gave the desired compound1.5 (244 mg) as an off-white solid.

Example 1, Step-D4-Amino-1-(2-C-methyl-β-D-ribofuranosyl)-3-[2-methoxycarbonyl)ethenyl]-1H-pyrazolo[3,4-d]pyrimidine(1.6)

To a solution of compound 1.5 from Step-C (392 mg, 0.54 mmol) in DMF (10mL) was added CuI (44 mg, 0.23 mmol), methyl acrylate (2.0 mL, 23.23mmol), triethylamine (0.332 mL, 2.36 mmol) andtetrakis(triphenylphosphine)palladium[0] (133 mg, 0.12 mmol), heated at70° C. under Ar. The reaction mixture was heated at 70° C. for 36 hr.After this time, further CuI (44 mg, 0.23 mmol), methyl acrylate (2.0mL, 23.23 mmol), triethylamine (0.332 mL, 2.36 mmol) andtetrakis(triphenylphosphine)palladium [0] (133 mg, 0.12 mmol) were addedand the mixture was heated at 70° C. for a further 6 hr. Then thereaction mixture was cooled to room temperature and 8 mL of 1/1MeOH/CH₂Cl₂ was added. 100 mg of Dowex 1×2-100 Bicarb form was added,the suspension stirred at room temperature for 45 min then filtered. Theresin was washed with 5×10 mL MeOH/CH₂Cl₂: 1/1, the solvents wereevaporated and the residual DMF was finally evaporated by azeotropicevaporation with toluene (2×10 mL). The residue was redissolved in MeOHand pre-adsorbed on silica gel. Chromatographic purification on silicagel using 4-4.5% MeOH in CH₂Cl₂ as eluent gave desired ester 1.6 (216mg, yield 52%).

Example 1, Step-E2-(2-C-methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-1,2,3,5,6-pentaazabenzo[cd]azulene-7-one(1.7)

A solution of ester 1.6 (208.3 mg, 0.57 mmol) in 0.1M NaOCH₃ in MeOH washeated at reflux for 3 h, cooled to 0° C. and Dowex 50×100 (acidic form)was added until the solution pH became neutral. The reaction mixture wasfiltered and the resin was washed with MeOH. The solvent was evaporated,and the residue was purified using silica gel column chromatographyusing 4 to 4.5% MeOH in CH₂Cl₂ as eluent to give the desired compound1.7 (26.5 mg) as an off-white solid.

¹H NMR (DMSO-d₆) d 11.6 (s, NH, 1H), 8.56 (s, H-4, 1H), 7.31 (d, J 12Hz, CH, 1H), 6.28 (d, J 12 Hz, CH, 1H), 6.15 (s, H-1′, 1H), 5.28 (s,2′-OH, 1H), 5.14 (d, J 6.9 Hz, 3′-OH, 1H), 4.65 (t, J 5.7 Hz, 5′-OH,1H), 3.95-4.09 (m, H-3′, H-4′, 2H), 3.62-3.69 (m, H-5′, 2H), 0.83 (s,CH₃, 3H). MS m/z 332 (M-H)⁺.

Example 22-(2-C-methyl-β-D-ribofuranosyl)-2,6,8,9-tetrahydro-7-oxa-2,3,5,6-tetraazabenzo[cd]azulene(2.9)

Bromination (NBS/THF) of starting pyrrolo pyrimidine 2.1 affordedbromopyrrolo pyrimidine 2.2 in 63% yield. Treatment of 2.2 withn-butyillithum in THF at −78° C. resulted in a selective lithio-bromoexchange to yield an intermediate which on treatment with(2-bromoethoxy) tert-butyldimethylsilane (−30° C./5 h) deliveredpyrimidine 2.3 in 43% yield along with recovered starting pyrimidine 2.2(30%). When the reaction was repeated on large scale (7.0 g of 2.2),significant improvement was achieved (−20° C./16 h) and pyrimidine 2.3was isolated in 55% yield (5.5 g). Stereoselective glycosylation of 2.3with bromo sugar 2.4 (freshly prepared form commercially available3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-1-O-methyl-α-D-ribofuranoseusing HBr in acetic acid/CH₂Cl₂) with 85% KOH/TDA-1(tris[2-(2-methoxyethoxy)ethyl]amine afforded nucleoside 2.5 in 26%isolated yield. Removal of the TBDMS group of 2.5 withtetrabutylammonium fluoride/THF gave diol 2.6. Compound 2.6 underwentMitsunobu coupling with N-hydroxyphthalimide to give the correspondingphthalimidooxyethyl nucleoside 2.7 in 88% yield. 2.7 was treated withanhydrous H₂NNH₂ to remove the phthaloyl group and this free amino oxyintermediate (crude) 2.8 was heated in EtOH to give 2.8. Removal of thedichlorobenzyl groups of 2.8 by using BCl₃ in CH₂Cl₂ delivered tricyclicnucleoside 2.9.

Example 2, Step-A 5-Bromo-4-chloropyrrolo[2,3-d]pyrimidine (2.2)

To solution of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was preparedaccording to a literature procedure (Townsend, L. B. et al., J. Med.Chem. 1988, 31, 2086-2092).

Example 2, Step-B5-[2-(tert-Butyldimethylsiloxy)ethyl]-4-chloro-7H-pyrrolo[2,3-pyrimidine(2.3)

A suspension of compound 2.2 from Step A (2.0 g, 8.6 mmol) in THF (40.0mL) was cooled to −78° C. under an argon atmosphere. n-BuLi (1.6M inhexanes, 13.4 mmol) was then added over 1.0 hr via syringe. A suspensionformed and (2-bromoethoxy)-tert-butyldimethylsilane (7.4 mL, 34.4 mmol)was added via syringe while maintaining the reaction mixture at −78° C.The reaction mixture was allowed to slowly reach −30° C. and stirred for2 h, then −30 to −10° C. for 1 h, and −10 to 0° C. for 1 hr. Thereaction mixture became dark brown in color, was treated with NH₄Cl,CH₂Cl₂ and water. The reaction mixture was extracted with CH₂Cl₂ and theextracts were dried over Na₂SO₄, filtered and evaporated. The beigeresidue was purified by flash column chromatography using 23% EtOAc inhexanes as eluent to give the desired compound 2.3 (1.16 g, 43%) as awhite solid. (Brown, D. M. et al., J. Chem. Soc. PT-1, 3565-3570.)

Example 2, Step-C5-[2-(tert-Butyldimethylsiloxy)ethyl]-4-chloro-7-[2-C-methyl-3,5-bis-O-(2,4-dichlorophenylmethyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(2.5)

Compound 2.3 (2.5 g, 8.00 mmol) was suspended in CH₃CN (50 mL), andpowdered 85% KOH, (1.3 g, 19.73 mmol) followed by TDA -1(tris[2-(2-methoxyethoxy)ethyl]amine) (0.2 mL, 0.62 mmol) were added.After stirring at room temperature for 10 min, a freshly preparedsolution of bromo sugar 2.4 (prepared from commercially available3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-1-O-methyl-α-D-ribofuranose;(i) Helv. Chim. Acta, 1995, 78, 486; (ii) WO 02/057287, 2002) (10.1mmol) in anhydrous acetonitrile (50 mL) was added via cannula, and thereaction stirred for 24 hr at room temperature then cooled in anice/water bath and treated with CH₂Cl₂ (100 mL) and water (80 mL). Theaqueous material was extracted three times with CH₂Cl₂, the combinedorganic layers were dried over Na₂SO₄, filtered and evaporated The crudeproduct was purified on a silica gel column using 20% ethylacetate/hexanes as eluent to give the desired nucleoside 2.5 (1.03 g,13%). Further elution with 25% EtOAc/hexanes as eluent gave a mixture ofthe desired nucleoside 2.5 and starting base 2.3 (350 mg).

Example 2, Step-D4-Chloro-7-[2-C-methyl-3,5-bis-O-(2,4-dichlorophenylmethyl)-β-D-ribofuranosyl]-5-(2-hydroxyethyl)-7H-pyrrolo[2,3-d]pyrimidine(2.6)

To a solution of compound 2.5 (1.14 g, 1.47 mmol) in THF (30 mL) wasadded a 1.0M solution of tetrabutylammonium fluoride in THF (2.2 mmol)at room temperature. The colorless solution was stirred for 5 hr at roomtemperature and then diluted by addition of 10 mL of CH₂Cl₂ and 10 mL ofbrine. The aqueous layer was extracted three times with CH₂Cl₂, driedover Na₂SO₄, filtered and evaporated. The residue obtained was purifiedby silica gel column chromatography using 1-1.5% MeOH in CH₂Cl₂ aseluent to give the desired compound 2.6 (900 mg, 88%) as a white solid.

Example 2, Step-E4-Chloro-7-[2-C-methyl-3,5-bis-O-(2,4-dichlorophenylmethyl)-β-D-ribofuranosyl]-5-(2-phthalimidooxyethyl)-7H-pyrrolo[2,3-d]pyrimidine(2.7)

To a solution of the compound 2.6 (359.0 mg, 0.54 mmol) in THF (10 mL)were added triphenylphosphine (215.0 mg, 0.82 mmol) andN-hydroxyphthalimide (132.0 mg, 0.0.81 mmol) followed bydiethylazodicarboxylate (DEAD) (153 μL, 0.88 mmol), and the solution wasstirred overnight at room temperature. The reaction mixture was dilutedby adding 10 mL of CH₂Cl₂ and 10 mL of water. The aqueous layer wasextracted three times with CH₂Cl₂, dried over Na₂SO₄, filtered andevaporated. The product was purified by flash column chromatographyusing 15-20% EtOAc in hexanes as eluent gave the desired compound 2.7(369 mg, 85%) as a white solid.

Example 2, Step-F2-(2-C-methyl-3,5-bis-O-(2,4-dichlorophenylmethyl)-β-D-ribofuranosyl)-2,6,8,9-tetrahydro-7-oxa-2,3,5,6-tetraazabenzo[cd]azulene(2.8)

To a suspension of compound 2.7 (880.0 mg, 1.09 mmol) in acetonitrile(60 mL) was added anhydrous hydrazine (38 μl, 1.19 mmol), and thesolution was stirred for 2 hr at room temperature. No starting materialleft as judged by the tlc. The white precipitate (phthalic hydrazide)was filtered off and washed with anhydrous acetonitrile, and thensolution was evaporated to dryness. The crude reaction product was driedunder high vacuum to give 864 mg of a white solid. The resulting freeaminooxy intermediate was redissolved in anhydrous EtOH (50 mL) and thesolution was heated at reflux for 2 d. After evaporation, reactionmixture was purified by silicagel column chromatography using CH₂Cl₂ to2% MeOH in CH₂Cl₂ to give desired compound 2.8 (466 mg) as a white foam.

Example 2, Step-G2-(2-C-methyl-β-D-ribofuranosyl)-2,6,8,9-tetrahydro-7-oxa-2,3,5,6-tetraazabenzo[cd]azulene(2.9)

To a solution of compound 2.8 (128.0 mg, 0.20 mmol) in CH₂Cl₂ (25 mL) at−78° C., was added a 1.0M solution of BCl₃ in CH₂Cl₂ (2.0 mL, 2.0 mmol)dropwise via syringe. The mixture was stirred at −78° C. for 1.5 h, thenat −35° C. to −40° C. for 2.5 hr. The reaction was quenched with MeOH(8.0 ml) and the solvents were evaporated. The resulting crude productwas purified by flash column chromatography over silica gel using 5%MeOH in CH₂Cl₂ as eluent to give the title compound 2.9 (59.2 mg) as awhite foam.

¹H NMR (DMSO-d₆) d 10.62 (s, NH, 1H), 8.19 (s, H-2, 1H), 7.47 (s, H-6,1H), 6.15 (s, H-1′, 1H), 5.09 (br s, 2′-OH, 3′-OH, 5′-OH, 3H), 4.29 (brs, OCH₂CH₂, 2H), 3.63-3.96 (m, H-3′, H-4′, H5′, 4H), 2.91-2.96 (m,OCH₂CH₂, 2H), 0.70 (s, CH₃, 3H). MS m/z 381 (M+CH₃COO)⁻.

Example 32-(β-D-ribofuranosyl)-2,6-dihydro-7H-1,2,3,5,6-pentaazabenzo[cd]azulene-7-one(3.3)

Example 3, Step-A4-Amino-1-(β-D-ribofuranosyl)-3-[2-methoxycarbonyl)ethenyl]-1H-pyrazolo[3,4-d]pyrimidine(3.2)

To a solution of compound 3.1* (300 mg, 0.76 mmol) in DMF (10 mL) wasadded CuI (29 mg, 0.15 mmol), methylacrylate (1.3 mL, 15.1 mmol),triethylamine (1.3 mL, 3.09 mmol) andtetrakis(triphenylphosphine)palladium[0] (88 mg, 0.08 mmol) and themixture heated at 70° C. for 36 hr under argon. After this time,additional CuI, methylacrylate, triethylamine and Pd catalyst wereadded, and the dark brown reaction mixture was heated for a further 6hr. Then the reaction mixture was cooled to room temperature and 8 mL of1/1 MeOH/CH₂Cl₂ was added. 100 mg Dowex 1×2-100 Bicarb form, was addedand the mixture stirred for 45 min at room temperature then filtered.The resin was washed with 2×10 mL MeOH/CH₂Cl₂:1/1 and the solventsevaporated. Chromatographic purification on silica gel using 6-7% MeOHin CH₂Cl₂ gave the desired compound 3.2 (120 mg) as a light yellowsolid.*J. Med. Chem. 1993, 36, 3424-3430.

Example 3, Step-B2-(β-D-ribofuranosyl)-2,6-dihydro-7H-1,2,3,5,6-pentaazabenzo[cd]azulene-7-one(3.3)

A solution of compound 3.2 (110 mg, 0.31 mmol) in 0.1M NaOCH₃ in MeOHwas heated at reflux for 3 h, cooled to 0° C. and treated with Dowex50×100 (acidic form) until the pH of the mixture became neutral. Thereaction contents were filtered and the resin was washed withMeOH/CH₂Cl₂ (1:1). The solvents were evaporated and the residue obtainedwas purified by flash column chromatography using 4-4.5% MeOH in CH₂Cl₂as eluent to give the title compound 3.3 (10.7 mg) as a white solid. ¹HNMR (DMSO-d₆) d 11.6 (s, NH, 1H), 8.58 (s, H-4, 1H), 7.35 (d, J 12 Hz,CH, 1H), 6.31 (d, J 12 Hz, CH, 1H), 6.11 (d, J 4.5 Hz, H-1′, 1H), 5.46(d, J 5.7 Hz, 2′-OH, 1H), 5.21 (d, J 3.9 Hz, 3′-OH, 1H), 4.80 (t, J 5.1Hz, 5′-OH, 1H), 4.62-4.64 (m, H-2′, 1H), 3.92-3.94 (m, H-3′, 1H),3.44-3.61 (m, H-4′, H5′, 2H), 0.83 (s, CH₃, 3H).

Example 42-(2-C-methyl-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene(4.6)

Treatment of 2.2 with n-butyllithium in THF at −78° C. resulted in aselective halogen-metal exchange to yield an intermediate which ontreatment with (3-bromopropoxy) tert-butyldimethylsilane (−30° C./5 h)afforded pyrimidine 4.1 in 36% yield along with the recovery of startingpyrimidine (30%). When the reaction was repeated on large scale(starting 7.0 g of 2.2), significant improvement was achieved (−20°C./16 h) and pyrimidine 4.1 was isolated in 55% yield. Stereoselectiveglycosylation of 4.1 with bromo sugar 2.4 in the presence of KOH/TDA-1afforded nucleoside 4.2 in 17% isolated yield. Removal of the TBDMSprotecting group of 4.2 allowed Mitsunobu coupling with phthalimide togive the corresponding phthalimido derivative 4.4 in quantitative yield.Phthalimide cleavage using hydrazine in different solvents failed toproduce primary amine and it was found that an alternative reactioncondition using ethylenediamine/ethanol resulted in phthalimide cleavagefollowed by in situ cyclization of the free amino intermediate to giveprotected tricyclic 4.5 in 56% yield. Deprotection of the dichlorobenzylgroups of 4.5 using BCl₃ in CH₂Cl₂/−78° C. to −30° C. delivered thetarget tricyclic nucleoside 4.6 in 84% isolated yield after purificationby silica gel column chromatography.

Example 4, Step-A5-[2-(tert-Butyldimethylsiloxy)propyl]-4-chloro-7H-pyrrolo[2,3-d]pyrimidine(4.1)

A suspension of compound 2.2 (10.0 g, 43.0 mmol) in THF (140.0 mL) wascooled to −78° C. under an argon atmosphere and n-BuLi (1.6M in hexanes,67.3 mmol) was then added via syringe over 1.5 hr. The resultingsuspension was stirred for the next 30 min at −78° C. then(3-bromopropoxy)-tert-butyldimethylsilane (21.4 mL, 172.0 mmol) wasslowly added via syringe at −78° C. over 1 hr. The reaction mixture wasallowed to slowly reach −30° C. and stirred for the next 2 h, and −30 to10° C. for 1 h, and −10 to 0° for 1 hr. The reaction mixture became darkbrown in color, and was kept at 4° C. overnight. The reaction wasquenched by adding aqueous NH₄Cl (100 mL), and diluted with CH₂Cl₂ (600mL) and water (120 mL) then extracted with CH₂Cl₂. The combined organiclayers were dried over Na₂SO₄, filtered and evaporated. The beigecolored residue was purified by flash column chromatography using 25%EtOAc in hexanes as eluent to give the desired compound 4.1 (5.5 g) as awhite solid.

Example 4, Step-B5-[2-(tert-Butyldimethylsiloxy)propyl]-4-chloro-7-(2-C-methyl-3,5-bis-O-(2,4-dichlorophenylmethyl)-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(4.2)

Compound 4.1 (5.24 g, 16.0 mmol) was suspended in CH₃CN (120 mL) andpowdered 85% KOH ((2.63 g, 40.0 mmol) was added followed by TDA-1(tris[2-(2-methoxyethoxy)ethyl]amine (0.4 mL, 1.24 mmol). After stirringat room temperature for 30 min, a freshly prepared solution of bromosugar 2.4 (20.0 mmol) in anhydrous acetonitrile (120 mL) was added viacannula, and the mixture stirred for 2 days at room temperature, cooledin an ice bath and treated with CH₂Cl₂ (200 mL) and water (100 mL). Theaqueous material was extracted three times with CH₂Cl₂ and the combinedorganic extracts were dried over Na₂SO₄, filtered and evaporated. Thecrude product was purified on a silica gel column using 15% ethylacetate/hexanes as eluent to give desired compound 4.2 (2.6 g) as alight yellow solid.

Example 4, Step-C4-Chloro-7-[2-C-methyl-3,5-bis-O-(2,4-dichlorophenylmethyl-β-D-ribofuranosyl]-5-(2-hydroxypropyl)-7H-pyrrolo[2,3-d]pyrimidine(4.3)

To a solution of compound 4.2 (800 mg, 1.01 mmol) in THF (20 mL) wasadded a 1.0 M solution of tetrabutylammonium fluoride in THF (1.5 mmol)at room temperature. The colorless solution was stirred for 4 hr at roomtemperature and then diluted by addition of 150 mL of CH₂Cl₂ and water(50 mL). The aqueous layers were extracted three times with CH₂Cl₂, andthen dried over Na₂SO₄, filtered and evaporated. Purification by silicagel column chromatography using 1-2% MeOH in CH₂Cl₂ as eluent gave thedesired compound 4.3 (460 mg, 67%) as a white solid.

Example 4, Step-D4-Chloro-7-[2-C-methyl-3,5-bis-O-(2,4-dichlorophenylmethyl)-β-D-ribofuranosyl]-5-(2-phthalimidoproyl)-7H-pyrrolo[2,3-d]pyrimidine(4.4)

To a solution of compound 4.3 (1.28 g, 1.89 mmol) in THF (70 mL) wereadded triphenylphosphine (648 mg, 2.47 mmol) and phthalimide (364.0 mg,2.47 mmol) followed by DEAD (440 μL, 2.53 mmol), and the solution wasstirred for overnight at room temperature. The reaction mixture wasdiluted by adding 100 mL of CH₂Cl₂ and water (100 mL) and the aqueouslayers extracted three times with CH₂Cl₂, dried over Na₂SO₄, filteredand evaporated. The residue obtained was purified by flash columnchromatography using 20-30% EtOAc in hexanes as eluent to give thedesired compound 4.4 (1.5 g, 100%) as a white solid.

Example 4, Step-E2-[2-C-methyl-3,5-bis-O-(2,4-dichlorophenylmethyl)-β-D-ribofuranosyl]-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene(4.5)

To a solution of compound 4.4 (161 mg, 0.2 mmol) in absolute EtOH (15mL) was added ethylenediamine (24 μL, 0.4 mmol) and the mixture wasstirred at 50° C. for 2 days. The solvents were evaporated and theresidue was purified by column chromatography using 2-3% MeOH in CH₂Cl₂as eluent to give the desired compound 4.5 (70.7 mg, 55%) as anoff-white foam.

Example 4, Step-F2-(2-C-methyl-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene(4.6)

To a solution of compound 4.5 (68.0 mg, 0.1 mmol) in CH₂Cl₂ (10 mL) at−78° C. was added 1.0 M solution of BCl₃ in CH₂Cl₂ (1.07 mmol) dropwisevia syringe. The mixture was stirred at −78° C. for 1.5 h, then for 3 hrat −35° C. to −40° C. The reaction was quenched by adding MeOH (6.0 mL),the solvents were evaporated and the resulting residue was purified byflash column chromatography over silica gel using 6-7% MeOH in CH₂Cl₂ aseluent to give title compound 4.6 (31.8 mg) as a white foam.

¹H NMR (DMSO-d₆) d 7.99 (s, H-2, 1H), 7.5 (s, NH, 1H), 7.23 (s, H-7,1H), 6.12 (s, H-1′, 1H), 3.82-3.94 (m, H-3′, H-4′, H-5′, 4H), 2.78 (m,NCH₂CH₂CH₂, 2H), 1.9 (m, NCH₂CH₂CH₂, 4H), 0.70 (s, CH₃, 3H). MS m/z 379(M+CH₃COO)⁻

Example 52-(2-O-Methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(5.9)

Selective 2′-O-methylation of tricyclic nucleosides was sought byintroduction of selective and simultaneous protection of 3′ and5′-hydroxyl groups in 5.1 through a 3′,5′-O-tetraisopropyldisiloxanebridge, followed by methylation and removal of 3′,5′-OH protection toafford 5.4.

Reaction of commercially available 6-chlorotubercidine 5.1 withTIPDSCl₂/imidazole in DMF/room temperature/overnight gave 5′,3′-O TIPDSprotected compound 5.2 in 75% yield. Methylation of 5.2 in DMF usingNaH/MeI/4 h/0° C. gave 2′OCH₃ compound 5.3 in 35% yield (2 steps). Itwas found that by decreasing the reaction time from 4 hr to 1 h,compound 5.3 could be isolated in 68% yield. Removal of the TIPDS of 5.3was accomplished by using 4 equivalent of 1.0M tetrabutylammoniumfluoride in THF at 0° C./1 hr to give 5.4. Intermediate 5.4 wasacetylated using Ac₂O/pyridine to give 5.5 in quantitative yield.Iodination of 5.5 using ICl in CH₂Cl₂ gave the iodo compound 5.6 in 66%yield and subsequent amination of 5.6 with methanolic ammonia 120° C./16h provided 5.7 in 89% isolated yield. Stille coupling of 5.7 using(Z)-methyl-3-(tributylstannyl)acrylate provided 5.8 (Z isomer) in 27%yield and the Stille coupling product 5.8, when subjected to cyclizationusing DBU/dioxane afforded target tricyclic nucleoside 5.9 in 48% yield.

Example 5, Step-A4-Chloro-7-[3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]-pyrimidine(5.2)

To a solution of commercially available 6-chlorotubercidine 5.1 (3.0 g,10.5 mmol) in DMF (140 mL) was added imidazole (3.6 g, 52.9 mmol), andTIPDSiCl₂ (1.2 eq) (4.0 mL, 12.5 mmol) at room temperature under argon.The reaction mixture was stirred for 16 h at room temperature and thenquenched by adding 20 mL of EtOH. The solvents were evaporated, water(100 mL) was added and the white suspension was extracted, with CH₂Cl₂.The organic extracts were dried over Na₂SO₄, filtered, evaporated andthe residue purified by flash column chromatography using gradient 5-7%EtOAc in hexanes to give the desired compound 5.2 (4.14 g, 75%) as aglassy solid.

Example 5, Step-B4-Chloro-7-[2-O-methyl-3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(5.3)

To a solution of compound 5.2 (2.0 g, 3.79 mmol) in DMF (45.0 mL) at 0°C., was added methyl iodide (1.88 mL, 15.23 mmol) followed by NaH (inone portion) (228 mg, 5.7 mmol, 60% suspension). The resulting reactionmixture was stirred for 1 hr at 0° C. and then quenched with anhydrousethanol (20 mL) and diluted with 100 mL of CH₂Cl₂. The diluted reactionmixture was washed with water and the organic phase was dried overNa₂SO₄, filtered, evaporated and coevaporated three times with toluene.The residue obtained was purified by flash column chromatography using agradient of 5-7% EtOAc in hexanes and afforded the desired 2′-O-methylnucleoside 5.3 (1.4 g, 68%)

Example 5, Step-C4-Chloro-7-(2-O-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(5.4)

To a solution of compound 5.3 (3.62 g, 6.66 mmol) in THF (100 mL), wasadded a 1.0 M solution of tetrabutylammonium fluoride in THF (26.0 mmol)at room temperature. The colorless solution was stirred for 1 hr at roomtemperature and then diluted by adding 150 mL of CH₂Cl₂ and water 50 mL.The aqueous portion was extracted three times with CH₂Cl₂, dried overNa₂SO₄, filtered and evaporated. Purification by silica gel columnchromatography using pure CH₂Cl₂ to 2% MeOH in CH₂Cl₂ as eluent gave thedesired compound 5.4 (1.24 g, 59%) as a colorless oil.

Example 5, Step-D4-Chloro-7-(2-O-methyl-3,5-di-O-acetyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(5.5)

To a solution of compound 5.4 (2.44 mmol) pyridine (40 mL), was addedacetic anhydride (1.23 mL, 8.44 mmol) via syringe and the solutionstirred overnight at room temperature. The solvents were evaporated invacuo, the residue was dissolved in CH₂Cl₂ and the solution was washedwith water then dried over Na₂SO₄, filtered, evaporated andco-evaporated with toluene three times. The residue obtained waspurified by dash column chromatography using a gradient of pure CH₂Cl₂to 2.5% MeOH in CH₂Cl₂ to give the desired compound 5.5 (1.26 g) as acolorless oil.

Example 5, Step-E4-Chloro-5-iodo-7-(2-O-methyl-3,5-di-O-acetyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(5.6)

To a solution of compound 5.5 (1.25 g, 3.26 mmol) in CH₂Cl₂ (80 mL), a1.0 M solution of ICl in CH₂Cl₂ (8.12 mmol) was added at roomtemperature and the resulting dark brown solution was stirred for 4 hrat room temperature. The solvents were evaporated in vacuo at 25° C.-35°C. and the residue was dried under high vacuum for 30 min. A light brownsticky material was obtained which was purified by flash columnchromatography using gradient of pure CH₂Cl₂ to 1.5% MeOH in CH₂Cl₂ toafford the desired compound 5.6 (1.1 g, 66%) as a white solid.

Example 5, Step-F4-Amino-5-iodo-7-(2-O-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(5.7)

A solution of compound 5.6 (28.8 mg, 0.057 mmol) in MeOH (3 mL) wastransferred to a steel bomb, cooled to −50 to −60° C., and treated witha saturated solution of ammonia in MeOH (10 mL). The reactor was sealedand heated at 118° C. overnight. The reaction vessel was cooled (0-5°C.), opened carefully and the reaction mixture was evaporated todryness. The crude product was dissolved in MeOH, adsorbed onto silicagel and purified by flash column chromatography on silica gel using pureCH₂Cl₂ to 4% MeOH in CH₂Cl₂ as eluents to give the desired compound 5.7(20.7 mg) as an off white solid.

Example 5, Step-G4-Amino-7-(2-O-methyl-β-D-ribofuranosyl)-3-[2-methoxycarbonyl)ethenyl]-7H-pyrrolo[2,3-d]pyrimidine(5.8)

To a solution of compound 5.7 (156 mg, 0.38 mmol) in DMF (12.0 mL), wereadded (Z)-methyl-3-(tributylstannyl)acrylate (J. Am. Chem. Soc., 1993,115, 1619) (0.29 mL, 0.77 mmol) and CuI (14.6 mg, 0.08 mmol). Themixture was stirred for 10 min at room temperature and then Pd(PPh₃)₂Cl₂was added, and reaction the mixture was heated for 3.5 h at 70° C. underargon. The reaction mixture was cooled to room temperature and filteredthrough a celite pad. The celite pad was washed with 8 mL of 1/1MeOH/CH₂Cl₂. After filtration, the washings were combined and thesolvents were evaporated in vacuo. The residue was redissolved in MeOHand adsorbed onto silica gel and purified by flash column chromatographyusing 2.5% MeOH in CH₂Cl₂ as eluent to give the desired compound 5.8 (38mg, 27%) as a yellow solid.

Example 5, Step-H2-(2-O-Methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6tetraazabenz[cd]azulen-7-one (5.9)

To a solution of compound 5.8 (36 mg, 0.1 mmol) in 1,4-dioxane (6.0 mL),were added 3A molecular sieves followed by DBU (38 μL, 0.25 mmol). Thereaction mixture was stirred for 3.5 h at 110° C. and then cooled toroom temperature and filtered, the solvents were evaporated and theresidue purified by flash column chromatography using CH₂Cl₂ to 2% MeOHin CH₂Cl₂ as eluent to give title compound 5.9 (16 mg) as an off whitesolid.

¹H NMR (DMSO-d₆) d 10.69 (s, NH, 1H), 8.33 (s, H-4, 1H), 7.8 (s, H-1,1H), 7.04 (d, J 11.7 Hz, CH, 1H), 6.13 (d, J 6.0 Hz, H-1′, 1H), 5.67 (d,J 11.7 Hz, CH, 1H), 5.25 (d, J 5.4 Hz, 3′-OH, 1H), 5.12 (t, J 5.7 Hz,5′-OH, 1H ), 4.24-4.27 (m, H-4′, 1H), 4.12-4.15 (m, H-3′, 1H), 3.93 (m,H-2′, 1H), 3.55-3.61 (m, H-5′, 2H), 3.28 (s, OCH₃, 3H). MS m/z 331(M-H)⁺

Example 62-(2-C-Methyl-β-D-ribofuranosyl]-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(6.6)

The tricyclic nucleoside 6.7 was synthesized starting from4-chloro-1H-pyrrolo[2,3-d]pyrimidine 2.1. The nucleobase 2.1 was treatedwith N-iodosuccinimide in THF at room temperature for 4 hr to provide4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine 6.1. Then 6.1 was convertedto the corresponding sodium salt with sodium hydride in acetonitrile andreacted with bromo-sugar 2.4 ((i)Helv. Chim. Acta. 1995, 78, 486; (ii)WO 02/057287, 2002), to give nucleoside 6.2. which was directlyconverted to4-amino-5-iodo-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine6.3. A Stille coupling reaction between nucleoside 6.3 andZ-3-tributylstannylacrylate provided compound 6.4. The cyclization ofcompound 6.4 was accomplished by heating in DBU/dioxane overnight toafford the protected tricyclic nucleoside 6.5, which was then treatedwith boron trichloride in CH₂Cl₂ to produce nucleoside 6.6.

4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (6.1)

Compound 6.1 was prepared according to a published procedure (Townsend,L. B. et al., 1990, 33, 1982-1992.

Example 6, Step A4-Chloro-5-iodo-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(6.2)

Compound 2.4 was prepared according to the published procedure. ((i).Helv. Chim. Acta. 1995, 78, 486; (ii). WO 02/057287, 2002).

A solution of compound 2.4 (25 mmol) in anhydrous acetonitrile (90 mL)was added to a solution of the sodium salt of4-chloro-5-iodo-1H-pyrrolo[2,3-d]pyrimidine [generated in situ from4-chloro-5-iodo-1H-pyrrolo[2,3-d]pyrimidine 6.1 (6.99 g, 25 mmol) inanhydrous acetonitrile (250 mL), and NaH (60% in mineral oil, 1.0 g, 25mmol), after 4 hr of vigorous stirring at room temperature]. Thecombined mixture was stirred at room temperature for 24 h, and thenevaporated to dryness. The residue was suspended in water (250 mL) andextracted with CH₂Cl₂ (2×500 mL). The combined extracts were washed withbrine (300 mL), dried over Na₂SO₄, filtered and evaporated. The crudeproduct was purified on a silica gel column using ethyl acetate/hexanes(1/4-1/2) as the eluent. Fractions containing the product were combinedand evaporated in vacuo to give the desired product 6.2 (6.26 g, yield34%) as light yellow foam.

Example 6, Step B4-amino-5-iodo-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(6.3)

To a compound 6.2 (5 g, 6.7 mmol) in dioxane (100 mL) was added conc.NH₄OH (100 mL). The mixture was heated in a stainless steel autoclave at100° C. for 3 h, then cooled and evaporated in vacuo. The crude mixturewas dissolved in 100 mL of CH₂Cl₂ and washed with water and brine, driedover MgSO₄, filtered and concentrated to provide crude product. Thecrude product was then purified on a silica gel column with 5% MeOH inCH₂Cl₂ as eluent to give 4.32 g of 6.3 as white foam (yield 88%).

Example 6, Step C4-Amino-5-[2-(methoxycarbonyl)ethenyl]-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(6.4)

To a solution of compound 6.3 (1.25 g, 1.726 mmol) in 10 mL of anhydrousDMF was added Z-3-tributylstannylacrylate (1.1 mL, 2 eq.) (J. Am. Chem.Soc., 1993, 115, 1619), CuI (66 mg, 0.2 eq.) and PdCl₂(PPh₃)₂ (121 mg,0.1 eq.) at room temperature under the argon atmosphere. The reactionmixture was heated at 70° C. for 8 hr. Then the reaction mixture wascooled to room temperature and filtered through a celite pad. Thefiltrate was concentrated in vacuo to provide an orange-red oil as thecrude product which was purified on a silica gel column with 10-30% THFin CH₂Cl₂ as eluent to give 995 mg 6.4 as yellow foam (yield 81%).

Example 6, Step D2-[3,5-Bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-β-D-ribofuranosyl]-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(6.5)

To a solution of the compound 6.4 (1.15 g, 1.69 mmol) in 150 mL ofanhydrous dioxane under an argon atmosphere at room temperature wasadded DBU (630 μL, 2.5 eq.) and 1 g of 4A molecular sieves. The reactionmixture was heated at reflux for 16 hr then cooled to room temperatureand evaporated to dryness in vacuo. The residue was purified by silicagel chromatography with 1% MeOH in CH₂Cl₂ as eluent to provide 772 mg ofcompound 6.5 as a yellow solid (yield: 71%).

Example 6, Step E2-(2-C-Methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(6.6)

To a solution of the compound 6.5 (0.71 g, 1.1 mmol) in 30 mL ofanhydrous CH₂Cl₂ at −78° C. was added boron trichloride (1M solution inCH₂Cl₂, 11 mL, 11 mmol) dropwise. The mixture was stirred at −78° C. for2.5 h, then at −30° C. to −20° C. for 3 hr. The reaction was quenched byaddition of methanolic/CH₂Cl₂ (1:1) (5 mL) and the resulting mixturestirred at −15° C. for 30 min., then neutralized with aqueous ammonia at0° C. and stirred at room temperature for 15 min. The solid was filteredand washed with CH₂Cl₂/MeOH (1/1, 3×30 mL). Chromatography over silicagel using 5% MeOH in CH₂Cl₂ as eluent furnished the 282 mg of thedesired compound 6.6 as a yellow solid (yield: 77.8%). ¹H NMR (300 MHz,DMSO-d₆) δ 10.67 (br s 1H, NH), 8.32 (s, 1H, H-4), 7.86 (s, 1H, H-1),7.00 (d, J 12.6 Hz, H-8), 6.09 (s, 1H, H-1′), 5.63 (d, J 12.6 Hz, H-9),5.16 (m, 3H, 3×OH), 3.95 (m, 1H, H-3′), 3.88-3.56 (m, 3H, H-4′, 2×H-5′),0.73 (s, 3H, CH₃); ES MS: 391.5 (M+CH₃COO)⁻.

Example 72-(2-C-methyl-β-D-ribofuranosyl)-8,9-dihydro-3,5,6,9a-tetraazabenzo[cd]azulene(7.14)

Example 7, Step A 2-C-Methyl-D-ribofuranose (7.2)

To a suspension of 2-C-methyl-1,2,3,5-tetra-O-benzoyl-β-D-ribofuranose1.3, (50 g) in anhydrous MeOH (1000 mL) was added KCN (150 mg) and themixture was allowed to stir at room temperature under argon for 15 hrduring which time all the material dissolved in the solvent and thesolution became clear. The solvent was evaporated and the residue wasdried under vacuum to deliver 14.9 g of product 7.2.

Example 7, Step B 2,3-O-Isopropylidene-2-C-methyl-D-ribofuranose (7.31)

The material from Step A (7.2) (14.9 g, 86 mmol) was dissolved in dryacetone (1000 mL) and 1 mL of conc. H₂SO₄ was added. The mixture wasstirred at room temperature overnight and then carefully neutralizedwith saturated aqueous NaHCO₃ and the solvent evaporated. The residuewas dissolved in 500 mL ethyl acetate and washed with water (100 mL) andbrine (100 mL). The solution was dried over Na₂SO₄, filtered andevaporated. The residue was purified on a silica gel column using 2:1hexanes:EtOAc. Evaporation of the solvent afforded 14 g of the product7.3.

Example 7, Step C2,3-O-Isopropylidene-2-C-methyl-5-O-(triphenylmethyl)-D-ribofuranose(7.4).

To a solution of compound 7.3 (13.7 g, 67.4 mmol) in pyridine was addedchlorotriphenylmethane (23.5 g, 84.2 mmol) and the mixture was heated at60° C. for 15 hr under an argon atmosphere. The solvent was evaporatedand the residue was dissolved in ethyl acetate (200 mL) and washed withwater (150 mL), brine (150 mL) and dried over sodium sulphate. Afterfiltration and evaporation, the residue was loaded on a silica gelcolumn and eluted with 10:1 followed by 5:1 hexane:ethyl acetate.Evaporation of solvent under reduced pressure afforded 15 g of 7.4 as acolorless syrup.

Example 7, Step D3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-4-C-methyl-7-O-(triphenylmethyl)-D-allo-andD-altro-septononitrile (7.5).

To a suspension of NaH (95%, 1.23 g, 48.2 mmol) in dry DME (250 mL),diethyl cyanomethylenephosphonate (10.06 mL, 62 mmol) was added dropwiseat 0° C. over 15 minutes. After evolution of hydrogen ceased, compound7.4 (15 g, 33.5 mmol), in 250 mL dry DME was added to the resultingsolution over 30 minutes and then the mixture was stirred at roomtemperature for 2 hr. The reaction mixture was partitioned between ether(1000 mL) and water (1000 mL) and the aqueous layer was extracted with1000 mL ether. The combined ether extracts were washed with water, dried(Na₂SO₄) and filtered. The solvent was evaporated under reducedpressure, and the residue was purified by silica gel columnchromatography using 4:1 hexane:ethyl acetate as eluent. The solvent wasevaporated to give 7.5 as an off-white foam (14.8 g).

Example 7, Step E(2E)-3,6-Anhydro-2-deoxy-2-C-[(N,N-dimethylamino)methylidene]-4,5-O-isopropylidene-4-C-methyl-7-O-(triphenylmethyl)-D-allo-and D-altro-septononitrile (7.6).

To a solution of compound 7.5 (13.0 g, 27.68 mmol) in anhydrous CH₂Cl₂(60 mL) was added bis(dimethylamino)-tert-butoxymethane (22.87 mL,110.74 mmol) followed by dry dimethylformamide (2.3 mL). The mixture wasstirred at room temperature for 20 hr. After removal of solvents themixture was chromatographed on a silica gel column pre-treated withtriethylamine to deliver 11.4 g of 7.6 as a viscous oil.

Example 7, Step F(2E)-3,6-Anhydro-2-deoxy-2-C-(hydroxymethylidene)-4,5-O-isopropylidene-4-C-methyl-7-O-(triphenylmethyl)-D-allo-and D-altro-septononitrile (7.7).

To a solution of compound 7.6 (6 g, 11.44 mmol) in CHCl₃ (120 mL) wasadded a solution of TFA (3 mL) in water (200 mL) and the mixture wasstirred vigorously at room temperature for 16 hr. The organic layer wasseparated and washed with water and dried (Na₂SO₄) then filtered.Removal of solvent afforded 2-formyl nitrile (7.7) (2.5 g) in a formthat was used as such in the next step.

Example 7, Step G(2E)-3,6-Anhydro-2-deoxy-2-C-[(cyanomethylamino)methylidene]-4,5-O-isopropylidene-4-C-methyl-7-O-triphenylmethyl)-D-allo-and D-altro-septononitrile (7.8).

Crude 7.7 was dissolved in MeOH (25 mL) and 1.6 mL water was addedfollowed by aminoacetonitrile hydrochloride (0.78 g, 8.77 mmol) andsodium acetate trihydrate (1.3 g, 9.55 mmol). The mixture was stirred atroom temperature for 16 hr. After evaporation of the solvent, theresidue was dissolved in a minimum volume of CH₂Cl₂ and loaded on asilica gel column which was eluted with 30:1 CH₂Cl₂:MeOH. Evaporation ofsolvent under reduced pressure gave 2.2 g of product 7.8 as an anomericmixture.

Example 7, Step H3-Amino-2-cyano-4-(2,3-O-isopropylidene-2-C-methyl-5-O-triphenylmethyl-β-D-ribofuranosyl)-1H-pyrrole(7.9).

To a solution of compound 7.8 (8 g, 14.94 mmol) in CH₂Cl₂ (100 mL) at 0°C. was added 1,5-diaza[4.3.0]non-5-ene (DBN) (2.95 mL, 23.89 mmol)followed by ethyl chloroformate (2.35 mL, 23.89 mmol). The mixture waskept at 0-4° C. for 16 hr. Additional 2 mL of DBN was added and themixture was stirred at room temperature for 24 hr. After evaporation ofthe solvent the residue was purified on silica gel column using 4:1hexanes:EtOAc followed by 3:1 hexanes:EtOAc to obtain a major fractionas mixture of anomers. To this mixture of anomers (6.42 g, 10.56 mmol)in MeOH (100 mL) was added sodium carbonate (3 g) and stirred at roomtemperature for 1 hr. The insoluble residue was filtered off and thesolvent was removed under vacuum. The residue was purified on a silicagel column using 3:1 hexanes:EtOAc to obtain 5 g of product 7.9 as βanomer.

Example 7, Step I Part A.: 3-(tert-Butyldiphenylsilyloxy)propyl4-methylbenzenesulfonate

The title compound was prepared according to the procedure described byCaprio et al. Tetrahedron, 2001, 57, 4023-4034.

Part B:3-Amino-1-(3-tert-butyldiphenylsilyloxypropyl)-2-cyano-4-(2,3-O-isopropylidene-2-C-methyl-5-O-triphenylmethyl-β-D-ribofuranosyl)-1H-pyrrole(7.10)

To a solution of potassium t-butoxide (1M THF solution, 4.32 mL) in THF(30 mL) was added compound 7.9 (2 g, 3.73 mmol), followed by catalyticamount of 18-crown-6. The mixture was stirred under argon for 10 minutesduring which time the solution turned clear reddish brown. To thissolution was added the compound prepared according Part A (3.68 g, 7.47mmol) dissolved in 1.5 m of (anhydrous) dichloroethane. After 1 hour, afurther equivalent of tosylate was added and the mixture was stirred atroom temperature overnight. After evaporation of the solvent undervacuum, the residue dissolved in minimum CH₂Cl₂ was loaded on a silicagel column and eluted with 6:1 followed by 4:1 hexane:EtOAc. Evaporationof the solvent from the appropriate fractions afforded 1.5 g of product7.10.

Example 7, Step J4-Amino-5-(3-tert-butyldiphenylsilyloxypropyl)-7-(2,3-O-isopropylidene-2-C-methyl-5-O-triphenylmethyl-β-D-ribofuranosyl)-5H-pyrrolo[3,2-d]pyrimidine(7.11).

Compound 7.10 (600 mg, 0.72 mmol) and formamidine acetate (227.5 mg,2.61 mmol) were mixed with 20 mL ethyl alcohol and heated at refluxunder argon atmosphere for 8 hr. The solvent was evaporated and theresidue was loaded on a silica gel column and eluted with 20:1MeOH:CH₂Cl₂ to afford 555 mg product 7.11.

Example 7, Step K4-Amino-5-(3-hydroxypropyl)-7-(2,3-O-isopropylidene-2-C-methyl-5-O-triphenylmethyl-β-D-ribofuranosyl)-5H-pyrrolo[3,2-d]pyrimidine(7.12).

Compound 7.11 (555 mg, 0.65 mmol) was dissolved in anhydrous THF (10 mL)and 1.3 mL (1.3 mmol) of a 1M THF solution of tetrabutylammoniumfluoride was added. The mixture was stirred at room temperature for 2 hrand the solvent was then evaporated under reduced pressure and theresidue was loaded on a silica gel column and eluted with 15:1CH₂Cl₂:MeOH to give 281 mg of product 7.12.

Example 7, Step L2-(2,3-O-Isopropylidene-2-C-methyl-5-O-triphenylmethyl-β-D-ribofuranosyl)-8,9-dihydro-3,5,6,9a-tetraazabenzo[cd]azulene(7.13).

Compound 7.12 (160 mg, 0.26 mmol) was taken up in 2 mL CH₂Cl₂ and keptat 0° C. To this was added TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy,free radical, 1 mg) followed by an aqueous solution KBr (1 mg in 0.2 mLwater) and Aliquat®366 (6 μL) and NaOCl (0.35 M, 0.92 mL). The mixturewas stirred at 0° C. for 30 minutes. More CH₂Cl₂ was added and thereaction was washed with water (5 mL). The organic layer was dried overNa₂SO₄, filtered and evaporated under reduced pressure. The residue wasused in the next step without further purification.

Example 7, Step M2-(2-C-methyl-β-D-ribofuranosyl)-8,9-dihydro-3,5,6,9a-tetraazabenzo[cd]azulene(7.14).

Compound 7.13 was heated at 80° C. in 90% acetic acid for 12 hr. Thesolvent was evaporated and the crude product was purified by reversephase HPLC on a C18 column to afford 3 mg pure product 7.14. ¹H NMR(DMSO-d₆). δ 8.10 (s, 1H), 7.49 (s, 1H), 6.60 (s, 1H), 5.33 (s, 1H),4.57 (m, 3H), 4.03 (m, 1H), 3.72 (m, 1H), 3.51 (m, 2H), 2.91 (m, 2H),2.77 (m, 2H), 1.02 (s, 3H).

Example 82-(2-C-Methyl-β-D-ribofuranosyl]-2,6,8,9-tetrahydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(8.1)

A mixture of tricyclic nucleoside 6.6 (20 mg, 0.06 mmol) and 10% Pd/C(12.8 mg, 0.2 eq.) in 20 mL of MeOH under H₂ pressure (32 psi) wasshaken for 7 hr at room temperature. The mixture was filtered through0.45 μM filter. The combined filtrates were evaporated and purified on asilica gel column with 5% MeOH in CH₂Cl₂. 12 mg of pure compound 8.1 wasobtained (yield 60%). ¹H NMR (300 MHz, DMSO-d₆): δ 10.65 (br s, 1H, NH),8.37 (s, 1H, H-4), 7.48 (s, 1H, H-1), 6.12 (s, 1H, H-1′), 5.05 (m, 3H,3×OH), 3.89-3.12 (m, 4H, H-3′, H-4′, 2×H-5′), 2.86-2.74 (m, 4H, 2×H-8,2×H-9), 0.64 (s, 3H, CH₃); LCMS: ES-MS 393.6 (M+CH₃COO).

Example 92-(β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[c]azulen-7-one(9.4)

The nucleoside 9.2 was produced directly from nucleoside 9.1, which wasprepared starting from D-ribose (Bheemarao G. et al; J. Med. Chem. 2000,43, 2883-2893), using liquid ammonia at 85° C. overnight. Thepalladium[0] catalyzed cross-coupling reaction of compound 9.2, followedby the cyclization of compound 9.3 in 0.1 N NaOMe in MeOH, delivered thedesired tricyclic nucleoside 9.4.

Example 9, Step A4-amino-5-iodo-7-β-D-ribofurnaosyl-7H-pyrrolo[2,3-d]pyrimidine (9.2)

Compound 9.2 was prepared according to a published method (Bergstrom, D.E., et al., J. Org. Chem., 1981, 46, 1423).

Example 9, Step B4-Amino-5-[2-(methoxycarbonyl)ethenyl]-7-(2-C-methyl-β-D-ribofurnaosyl)-7H-pyrrolo[2,3-d]pyrimidine(9.3)

To a solution of compound 9.2 (300 mg, 0.76 mmol) in 10 mL of anhydrousDMF was added CuI (29 mg, 0.2 eq.), methyl acrylate (1.37 mL, 20 eq.),triethylamine (212 μL, 2 eq.) and Pd(PPh₃)₄ (88 mg, 0.1 eq.) at roomtemperature under an argon atmosphere. The reaction mixture was heatedat 70° C. for 24 hr. then cooled to room temperature and 20 mL of 1/1MeOH/CH₂Cl₂ was added. Then, 1.0 g Dowex 1×2-100 Bicarb form was addedand the suspension stirred at room temperature for 45 min. thenfiltered. The resin was washed with 5×20 mL MeOH/CH₂Cl₂:1/1, DMF wasfinally evaporated by co-evaporation with toluene (2×10 mL).Chromatographic column purification on silica gel (eluent:CH₂Cl₂/MeOH:90/10) gave 224 mg of final product 9.3 (yield 84%)

¹H NMR (CD₃OD) δ 8.11 (s, 1H, H-2), 8.00 (s, 1H, H-6), 7.96 (d, J 15.54Hz, 1H), 6.35 (d, J 15.54 Hz, H2″), 6.52 (d, 1H, H-1′), 4.43 (t, 1H,H-3′), 4.34-4.26 (m, 2H, H-4′, H-2′), 3.85-3.64 (m, 2H, 2×H-5′), 3.79(s, 3H, OCH₃).

Example 9, Step C2-(β-D-Ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(9.4)

A solution of compound 9.3 (140 mg, 0.4 mmoles) in 0.1 M NaOMe in MeOH(80 mL) was heated at 70° C. for 4 hr. The solution was cooled and thesolvent evaporated and the residue purified by silica gel column usingCH₂Cl₂/MeOH:90/10 as eluent to give the final product 9.4.

¹H NMR (CD₃OD) δ 8.32 (s, 1H, H-2), 7.86 (s, 1H, H-6), 7.07 (d, J 12 Hz,H-8), 6.09 (s, 1H, H-1′), 5.70 (d, J 12 Hz, H-9), 4.57 (m, 1H, H-2′),4.29 (m, 1H, H-3′), 4.12 (m, 1H, H-4′), 3.88-3.72 (m, 2H, 2×H-5′);ES-MS: 377.4 (M+CH₃COO)⁻.

Example 102-(3-Deoxy-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(10.6)

The nucleoside 5.1 was prepared starting from D-ribose ((i) Journal ofHeterocyclic Chemistry, 25(6), 1893-8, 1988; (ii) Helvetica ChimicaActa, 71(6), 1573-85, 1988), then converted to the 3′-deoxy nucleoside10.2 as follows: 4.0 equiv of α-acetoxyisobutyryl bromide was added to asuspension of nucleoside 5.1 in acetonitrile containing 1.1 equiv of H₂Oat room temperature followed by treatment with DOWEX OH⁻ resin in MeOHto afford a crystalline sample of4-methoxy-7-(2,3-anhydro-β-D-ribofurnaosyl)-7H-pyrrolo[2,3-d]pyrimidine10.1. The epoxide was treated with 4.0 equiv of LiEt₃BH in THF at roomtemperature to give the 3′-deoxynucleoside 10.2 in 50% combined yieldfor the two preceding steps. The 7-iodo group was introduced by reactingthe 3′-deoxynucleoside 10.2 with N-iodosuccinimide in DMF to givecompound 10.3 which, in turn, was treated with anhydrous liquid ammoniato provide compound 10.4. After the palladium[0] catalyzedcross-coupling reaction and cyclization to form the tricyclic target,the desired nucleoside 10.6 was obtained.

Example 10, Step A4-methoxy-7-(2,3-anhydro-β-D-ribofurnaosyl)-7H-pyrrolo[2,3-d]pyrimidine(10.1)

To a mixture of nucleoside 5.1 (250 mg, 0.875 mmol) in 12 mL ofacetonitrile were added H₂O/acetonitrile (1/9) (157 μL, 1 eq.) andα-acetoxyisobutyryl bromide (0.537 mL, 4 eq.). After 2 hr stirring atroom temperature, sat. NaHCO₃ (aq.) was added and the mixture wasextracted with ethyl acetate. The combined organic extracts were washedwith brine, dried over MgSO₄ and evaporated. The foamy residue wassuspended in MeOH and stirred overnight with Dowex OH⁻ previously washedwith anhydrous MeOH). The resin was filtered off, washed with MeOH andthe combined filtrates were evaporated to yield 225 mg of a pale yellowfoam 10.1, which was directly used in the next step without furtherpurification.

Example 10, Step B4-Methoxy-7-(3-deoxy-β-D-ribofurnaosyl)-7H-pyrrolo[2,3-d]pyrimidine(10.2)

Superhydride LiEt₃BH in 1M THF (8 mL, 10 eq.) was added dropwise to anice-cold deoxygenated (after 15 min purging with argon) solution ofanhydrous nucleoside 10.1 (218 mg, 0.8 mmol) in anhydrous THF (10 mL)under argon. The resulting mixture was stirred at 0° C. for 2 h, thenacidified cautiously and finally purged with argon for 1 hr. The residuewas purified on a silica gel column with 5% MeOH in CH₂Cl₂ to yield 117mg of target compound 10.2 as off-white solid (yield 55%).

Example 10, Step C4-Methoxy-5-iodo-7-(3-deoxy-β-D-ribofurnaosyl)-7H-pyrrolo[2,3-d]pyrimidine(10.3)

To a solution of nucleoside 10.2 (350 mg, 1.32 mmol) in DMF (10 mL) wasadded N-iodosuccinimide (327 mg, 1.1 eq.) at 0° C. The reaction mixturewas stirred at 0° C. under argon for 2 h, then warmed up to roomtemperature and stirred overnight. The reaction was quenched by additionof 4 mL of MeOH. The solution was evaporated to dryness, thenredissolved in CHCl₃, washed with sat. aq. NaHCO₃, Na₂SO₃ and water,then dried over MgSO₄. After evaporation, the residue was purified on asilica gel column using 0-3% MeOH in CH₂Cl₂ to provide 353 mg of purecompound 10.3 as white solid (yield: 68%).

Example 10, Step D4-Amino-5-iodo-7-(3-deoxy-β-D-ribofurnaosyl)-7H-pyrrolo[2,3-d]pyrimidine(10.4)

A mixture of compound 10.3 (50 mg, 0.128 mmol) and anhydrous liquidammonia (15 mL) was heated in a stainless steel autoclave at 120° C. 2days, then cooled and evaporated in vacuo. The residue was purified on asilica gel column with 3% MeOH in CH₂Cl₂ as eluent to give 30 mg of thecompound 10.4 as a white solid. (yield: 62%)

Example 10, Step E4-Amino-5-[2-(methoxycarbonyl)ethenyl]-7-(3-deoxy-β-D-ribofurnaosyl)-7H-pyrrolo[2,3-d]pyrimidine(10.5)

To a solution of compound 10.4 (50 mg, 0.132 mmol) in 2 mL of anhydrousDMF were added CuI (5 mg, 0.2 eq.), methyl acrylate (240 μL, 20 eq.),triethylamine (37 uL, 2 eq.) and Pd(PPh₃)₄ (15 mg, 0.1 eq.) at roomtemperature under an argon atmosphere. The reaction mixture was heatedat 70° C. for 48 hr. then cooled to room temperature and 20 mL of 1/1MeOH/CH₂Cl₂ was added. 100 mg Dowex 1×2-100 Bicarb form was then addedand the suspension was stirred at room temperature for 45 min. thenfiltered. The resin was washed with 3×10 mL MeOH/CH₂Cl₂:1/1, and thesolvent evaporated. DMF was finally evaporated by azeotropicco-evaporation with toluene (2×5 mL). The residue was purified bychromatographic column purification on silica gel (eluent:CH₂Cl₂/MeOH=95/5) to give 20 mg of final product 10.5 (yield 45%).

Example 10, Step F2-(3-Deoxy-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(10.6)

A solution of compound 10.5 (20 mg, 0.06 mmoles) in 0.1 M NaOMe in MeOH(12 mL) was heated at 70° C. for 4 hr. The solvent was evaporated andthe residue purified by silica gel column with CH₂Cl₂/MeOH=90/10) togive the final product 10.6.

¹H NMR (300 MHz, CD₃OD): δ 8.31 (s, 1H, ), 7.71 (s, 1H, ), 7.05 (d, J 12Hz), 6.05 (s, 1H, H-1′), 5.73 (d, J 12 Hz, 1H,), 3.87-3.66 (m, 4H, H-2′,H-4′ 2×H-5′), 2.31-2.09 (m, 1H, 2×H-3′); ES MS: 360.9 (M+CH₃COO).

Example 112-(2-C-Methyl-β-D-ribofuranosyl)-9-methyl-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(11.4)

Example 11, Step A4-Chloro-5-iodo-7-(2-C-methyl-β-D-ribofurnaosyl)-7H-pyrrolo[2,3-d]pyrimidine(11.1)

To a solution of the compound 6.3 (7.73 g, 10.39 mmoles) indichloromethane (200 mL) at −78° C. was added boron trichloride (1M indichloromethane, 104 mL, 104 mmol) dropwise. The mixture was stirred at−78° C. for 2.5 h, then at −30° C. to −20° C. for 3 hr. The reaction wasquenched by addition of methanolic/dichloromethane (1:1) (105 mL) andthe resulting mixture stirred at −15° C. for 30 min., then neutralizedwith aqueous ammonia at 0° C. and stirred at room temperature for 15min. The solid was filtered and washed with CH₂Cl₂/MeOH (1/1, 250 mL).The chromatography over silica gel using CH₂Cl₂ and CH₂Cl₂/MeOH (99/1 to90/10) gradient as the eluent to furnish the desired compound 11.1 (2.24g, yield 51%) as a colorless foam.

Example 11, Step B4-Amino-5-iodo-7-(2-C-methyl-β-D-ribofurnaosyl)-7H-pyrrolo[2,3-d]pyrimidine(11.2)

To the compound 11.1 (425 mg, 1 mmol) was added liquid ammonia (20 mL).The mixture was heated in a stainless steel autoclave at 85° C.overnight, then cooled and evaporated in vacuo. The crude mixture waspurified on a silica gel column with 5% methanol in dichloromethane aseluent to give the product 11.2 as a light yellow foam (400 mg, 100%yield).

Example 11, Step C4-Amino-5-[1-methyl-2-(methoxycaronyl)ethenyl]-7-(2-C-methyl-β-D-ribofurnaosyl)-7H-pyrrolo[2,3-d]pyrimidine(11.3)

To a solution of compound 11.2 (1 g, 2.46 mmol) in 20 mL of anhydrousDMF were added CuI (94 mg, 0.2 eq.), methyl crotonate (5.33 mL, 20 eq.),triethylamine (686 μL, 2 eq.) and Pd(PPh₃)₄ (285 mg, 0.1 eq.) at roomtemperature under an argon atmosphere. The reaction mixture was heatedat 70° C. for 24 hr. then cooled to room temperature and 100 mL of 1/1MeOH/CH₂Cl₂ was added. 2.0 g Dowex 1×2-100 Bicarb form was then addedand the suspension was stirred at room temperature for 45 min., thenfiltered. The resin was washed with 3×50 mL MeOH/CH₂Cl₂: 1/1. DMF wasfinally evaporated by azeotropic co-evaporation with toluene (2×5 mL).Chromatograph purification on silica gel (eluent: C_(H2)Cl₂/MeOH: 95/5)gave 463 mg of product 11.2 (yield: 50%).

Example 11, Step D2-(2-C-Methyl-β-D-ribofuranosyl)-9-methyl-2,6-dihydro-7H-2,3,5,6-tetraazabenz[cd]azulen-7-one(11.4)

A solution of compound 11.3 (33 mg, 0.087 mmoles) in 0.1 M NaOMe in MeOH(17 mL) was heated at 70° C. for 4 hr. The solution was evaporated andthe residue purified by silica gel chromatography using CH₂Cl₂/MeOH:95/5as eluent to provide 24 mg of final product 11.4 (yield 80%).

¹H NMR (300 MHz, DMSO-d₆) δ 10.66 (br. 1H, NH), 8.33 (s, 1H, H-4), 8.09(s, 1H, H-1), 6.10 (s, 1H, H-1′), 5.67 (s, 1H, H-8), 5.16 (m, 3H, 3×OH),4.05-3.65 (m, 4H, H-3′, H-4′ 2×H-5′), 2.11 (s, 3H, CH₃), 0.73 (s, 3H,CH₃); ESMS: 405.5 (M+CH₃COO).

Example 122-(2-C-Methyl-β-D-ribofuranosyl)-8-methyl-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(12.2)

Example 12, Step A4-amino-5-[2-methyl-2-(methoxycaronyl)ethenyl]-7-(2-C-methyl-β-D-ribofurnaosyl)-7H-pyrrolo[2,3-d]pyrimidine(12.1)

To a solution of compound 11.2 (200 mg, 0.492 mmol) in 4 mL of anhydrousDMF was added CuI (19 mg, 0.2 eq.), α-methyl methacrylate (1.06 mL, 20eq.), triethylamine (137 μL, 2 eq.) and Pd(PPh₃)₄ (57 mg, 0.1 eq.) atroom temperature under an argon atmosphere. The reaction mixture washeated at 70° C. for 24 hr. then cooled to room temperature and 100 mLof 1/1 MeOH/CH₂Cl₂ was added. 400 mg Dowex 1×2-100 Bicarb form was thenadded the suspension stirred at room temperature for 45 min., thenfiltered. The resin was washed with 3×10 mL MeOH/CH₂Cl₂:1/1. DMF wasfinally evaporated by azeotropic co-evaporation with toluene (2×5 mL).Chromatograph purification of the residue on silica gel (eluent:CH₂Cl₂MeOH:95/5) gave 100 mg of ester 12.1 (yield: 54%).

Example 12, Step B2-(2-C-Methyl-β-D-ribofuranosyl)-8-methyl-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(12.2)

A solution of compound 12.1 (30 mg, 0.079 mmoles) in 0.1 M NaOMe in MeOH(16 mL) was heated at 70° C. for 4 hr. The solution was evaporated andthe residue purified by silica gel column chromatography usingCH₂Cl₂/MeOH:95/5) to provide product 12.2.

¹H NMR (300 MHz, DMSO-d₆) δ 10.60 (br s. 1H, NH), 8.28 (s, 1H, H-4),7.73 (s, 1H, H-1), 7.09 (s, 1H, H-9), 6.07 (s, 1H, H-1′), 5.16 (m, 3H,3×OH), 3.93-3.63 (m, 4H, H-3′, H-4′ 2×H-5′), 1.97 (s, 3H, CH₃), 0.72 (s,3H, CH₃); ES MS: 405.3 (M+CH₃COO).

Example 132-(2-C-Methyl-β-D-ribofuranosyl)-9-methoxy-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[c,d]azulen-7-one(13.2)

Example 13, Step A4-Amino-5-[1-methoxy-2-(methoxycaronyl)ethenyl]-7-(2-Cmethyl-β-D-ribofurnaosyl)-7H-pyrrolo[2,3-d]pyrimidine (13.1)

To a solution of compound 11.2 (200 mg, 0.492 mmol) in 5 mL of anhydrousDMF were added CuI (19 mg, 0.2 eq.), E-3-methoxymethacrylate (1.06 mL,20 eq.), triethylamine (137 μL, 2 eq.) and Pd(PPh₃)₄ (57 mg, 0.1 eq.) atroom temperature under an argon atmosphere. The reaction mixture washeated at 70° C. for 24 hr. then cooled to room temperature and 100 mLof 1/1 MeOH/CH₂Cl₂ was added. 400 mg Dowex 1×2-100 Bicarb form was thenadded and the suspension stirred at room temperature for 45 min., thenfiltered. The resin was washed with 3×10 mL MeOH/CH₂Cl₂:1/1. DMF wasfinally evaporated by azeotropic co-evaporation with toluene (2×5 mL).Chromatographic purification of the residue on silica gel (eluent:CH₂Cl₂/MeOH:95/5) gave 87 mg of product 13.1 (yield: 45%).

Example 13, Step B2-(2-C-Methyl-β-D-ribofuranosyl)-9-methoxy-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(13.2)

A solution of compound 13.1 (30 mg, 0.076 mmoles) in 0.1 M NaOMe in MeOH(15 mL) was heated at 70° C. for 4 hr. The solution was evaporated andthe residue purified by silica gel column with CH₂Cl₂/MeOH:95/5) toprovide nucleoside 13.2.

¹H NMR (300 MHz, DMSO-d₆) δ 10.63 (br s 1H, NH), 8.35 (s, 1H, H-4), 8.06(s, 1H, H-1), 6.13 (s, 1H, H-1′), 5.30 (s, 1H, H-8), 5.22 (m, 3H, 3×OH),3.32 (s, 3H, OCH₃), 3.98-3.63 (m, 4H, H-3′, H-4′ 2×H-5′), 0.71 (s, 3H,CH₃); ES MS: 421.5 (M+CH₃COO⁻).

Example 142-(2-C-Methyl-β-D-ribofuranosyl)-8-bromo-9-methoxy-2,6,8,9-tetrahydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(14.1)

To a stirred solution of nucleoside 6.6 (10 mg, 0.030 mmol) in DMF (0.5mL) was added N-bromosuccinimide (11.25 mg, 2.10 eq.) at 0° C. underargon. The reaction mixture was stirred at 0° C. for 1 hr then quenchedwith MeOH (0.5 mL). The mixture was evaporated to dryness and theresidue was purified on a silica gel column with 5% MeOH in CH₂Cl₂ togive compound 14.1 as a mixture of diastereoisomers (8 mg, 65%). Theisolated compound was characterized by ¹H NMR, COSY, NOESY and LCMS. ¹HNMR (300 MHz, DMSO-d₆) δ 11.29 (s, 1H, NH), 8.54 (s, 1H, H-4), 8.00 (s,1H, H-1), 6.24, 6.21 (2s, 1H, H-1′), 5.22-5.08 (m, 4H, 3×OH, H-8),4.77-4.73 (m, 1H, H-9), 3.95-3.7 (m, 4H, H-3′, H-4′ 2×H-5′), 3.24, 3.21(2s, 3H, OCH₃), 0.68, 0.66 (2s, 3H, CH₃). ES MS: 501.7 (M+CH₃COO⁻).

Example 154-Amino-2-(2-C-methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulene-7-one(15.6)

The sodium salt of4-chloro-5-iodo-2-pivaloylamino-1H-pyrrolo[2,3-d]pyrimidine 15.1(prepared in situ using sodium hydride) was reacted with protected1-bromo-2-C-methyl-D-ribofuranose 2.4 (which was prepared with HBr/AcOHin CH₂Cl₂ from the corresponding 1-O-methyl analogue) to give theβ-anomer 15.2. Removal of dichlorophenymethyl protecting groups wasperformed using boron trichloride in CH₂Cl₂ to give the4-chloro-nucleoside 15.3. Further ammonolysis and deprotection atelevated temperature yielded 2,4-diamino nucleoside 15.4, which wasconverted under the Heck coupling conditions with methylacrylate intothe corresponding 5-methylpropenoate 15.5. This compound was convertedinto the target tetraazabenzo[cd]azulene nucleoside 15.6 via sodiummethoxide mediated ring closure.

Example 15, Step A4-Chloro-5-iodo-2-pivaloylamino-7-[3,5-bis-O-(2,4-dichlorophenymethyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(15.2)

A solution of 2.4 (36.7 mmol) in anhydrous acetonitrile (50 mL) wasadded to a solution of sodium salt of4-chloro-5-iodo-2-pivaloylamino-7H-pyrrolo[2,3-d]pyrimidine inacetonitrile [generated in situ from4-chloro-5-iodo-2-pivaloylamino-7H-pyrrolo[2,3-d]pyrimidine (Nucl. AcidRes. 1998 (26), 3350-3357) (20.87 g, 55.1 mmol) in anhydrousacetonitrile (1000 mL) and NaH (60% in mineral oil, 2.20 g, 55.1 mmol)after 4 hr of vigorous stirring at room temperature]. The combinedmixture was stirred at room temperature for 48 hr. The solids werefiltered then washed with acetonitrile (100 mL) and the combinedfiltrate evaporated to provide a viscous oil. Purification on a silicagel column, using hexanes/EtOAc gradient (15/1, 13/1, 11/1, 9/1, 7/1) asthe eluent, yielded the target compound as a colorless foam (7.02 g,23%).

Example 15, Step B4-Chloro-5-iodo-2-pivaloylamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(15.3)

To a solution of compound 15.2 (7.03 g, 8.43 mmol) in CH₂Cl₂ (200 mL) at−75° C. was added boron trichloride (1M in CH₂Cl₂; 83.4 mL, 83.4 mmol).The mixture was stirred at −75 to −70° C. for 2 hr and then at −30 to−20° C. for 3 hr. The reaction was quenched by addition of MeOH/CH₂Cl₂(1/1, 9 mL) and the resulting mixture stirred at −20 to −15° C. for 30min., then neutralized with aq. ammonia (28%, 35 mL) at 0° C. andstirred at room temperature for 10 min. The solid which separated wasfiltered and washed with MeOH/CH₂Cl₂ (1/1, 500 mL). The combinedfiltrates were evaporated and the residue was purified on a silica gelcolumn using CH₂Cl₂/MeOH (50/1, 40/1) as the eluents to furnish thetarget compound 15.3 as an off-white solid (2.93 g, 80%).

Example 15, Step C2,4-Diamino-5-iodo-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(15.4)

A mixture of the compound from Step B (2.92 g 5.6 mmol) and anhydrousliquid ammonia (50 mL) was heated in a stainless steel autoclave at 110°C. for 1 d, then cooled and the solvent evaporated. The residue wastreated with MeOH to yield 0.30 g of 15.4. The filtrate was evaporatedand purified on silica gel column with CH₂Cl₂/MeOH (20/1) to furnishadditional 1.52 g of the target compound (total yield 77%).

Example 15, Step D2,4-Diamino-5-[(E)-1-(methoxycarbonyl)-2-ethenyl]-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(15.5)

To a solution of the compound from Step C (1.54 g, 3.66 mmol) in DMF (35mL) were added copper iodide (139 mg, 0.73 mmol), methyl acrylate (6.6mL, 73.1 mmol), triethylamine (1.02 mL, 7.3 mmol), andtetrakis(triphenylphosphine)palladium [0] (422.5 mg, 0.37 mmol). Theresulting mixture was stirred at 70° C. for 10 h, then cooled to roomtemperature and diluted with MeOH/CH₂Cl₂ (1/1, 50 mL). Dowex HCO₃ ⁻ (3g) was added then and after 45 min of stirring, the resin was filteredoff, washed with CH₂Cl₂/MeOH (1/1, 150 mL) and the combined filtratesconcentrated. The residue was treated with MeOH and the catalyst, whichseparated, was filtered off. The evaporated filtrate was treated withMeOH again and the target compound, which separated, was filtered off(627 mg). The filtrate was concentrated in vacuo, and purified on asilica gel column using a CH₂Cl₂/MeOH gradient (50/1, 30/1, 20/1 and15/1) to furnish an additional 175 mg of compound 15.5 (total yield58%).

Example 15, Step E4-Amino-2-(2-C-methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulene-7-one(15.6)

A solution of the compound from Step D (578 mg, 1.52 mmol) in 0.1 NNaOMe/MeOH (250 mL) was heated at 60° C. for 12 hr and then neutralizedat room temperature with Dowex H⁺. The resin was filtered, washed withMeOH and the combined filtrates concentrated in vacuo. Purification on asilica gel column with CH₂Cl₂/MeOH (10/1 and 5/1) yielded the targetcompound 15.6 as a yellow solid (245 mg, 46%).

¹H-NMR (DMSO-d₆): δ 10.04 (br s, NH, 1H), 7.42 (s, 1H, H-1), 6.90 (d,H-9, J 11.7 Hz, 1H),. 6.26 (br, NH₂, 2H), 5.91 (s, H-1′, 1H), 5.56 (dd,J 11.7 Hz, J 1.6 Hz, H-8, 1H), 5.21 (br s, 3′-OH, 1H), 5.06 (t, J 4.8Hz, 5′-OH, 1H), 4.98 (s, 2′-OH, 1H), 3.75-3.88 (m, H-3′, H-4′, H-5′,3H), 3.62 (m, H-5′,1H), 0.78 (s, Me, 3H). MS m/z=406.5 (M+CH₃COO⁻).

Example 164-Fluoro-2-(2-C-methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulene-7-one(16.2)

Nucleoside 15.6 was converted with HF in pyridine and tert-butylnitriteat low temperature into the corresponding 4-fluoro analogue 16.2, after5′-O-tert-butyldimethylsilyl derivatization with tert-butyldimethylsilylchloride and imidazole in DMF.

Example 16, Step A4-Amino-2-(5-O-tert-butyldimethylsilyl-2-C-methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulene-7-one(16.1)

A mixture of4-amino-2-(2-C-methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulene-7-one(65 mg, 0.19 mmol) in DMF, tert-butyldimethylsilyl chloride (70 mg, 0.45mmol) and imidazole (61 mg, 0.90 mmol) was stirred overnight at roomtemperature and then concentrated in vacuo. The oily residue wasdissolved in CH₂Cl₂ (20 mL), washed with aq. HCl (0.1 N), sat.aq.NaHCO₃, water, brine and dried (Na₂SO₄). The evaporated residue waspurified on silica gel with hexanes/EtOAc (1/1)+0.5% Et₃N and EtOAc+0.5%Et₃N to yield the target compound 16.1 as colorless oil (38 mg, 44%).

Example 16, Step B4-Fluoro-2-(2-C-methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulene-7-one(16.2)

To a solution of HF/pyridine (1.5 mL) and pyridine (1 mL) at −25° C. wasadded a solution of 16.1 (30 mg, 0.07 mmol) in pyridine (0.5 mL)followed by tert-butylnitrite (15 μL, 0.13 mmol). Reaction mixture wasallowed to warm to −5° C., quenched with 6 N aq. NaOH and evaporated.The pale-yellow residue was triturated with MeOH, filtered andthoroughly washed with MeOH (100 mL combined filtrate). The evaporatedfiltrate was purified on a silica gel column with CH₂Cl₂/MeOH 20/1 togive the target compound 16.2 as a yellow solid (5 mg, 22%).

¹H-NMR (CD₃OD): δ 7.05 (d, H-9, J_(H8,H9)=11.7 Hz, 1H), 7.81 (s, 1H,H-1), 6.09 (s, H-1′, 1H), 5.78 (d, J_(H8,H9)11.7 Hz, H-8, 1H), 3.99-4.10(m, H-3′, H-4′, H-5′, 3H), 3.85 (dd, J_(gem) 12.9 Hz, J_(H5′,H4′) 3.5Hz, H-5′,1H), 0.92 (s, Me, 3H). ¹⁹F-NMR (CD₃OD): δ −52.58. MS m/z=409.6(M+CH₃COO⁻).

Example 177-Amino-2-(2-C-methyl-β-D-ribofuranosyl)-2H-2,3,5,6-tetraazabenzo[cd]azulene(17.2)

Nucleoside 11.2 was reacted with acrylonitrile under Heck-type couplingconditions. The nitrile 17.1 was converted into the target7-amino-tetraazabenzoazulen nucleoside 17.2 via sodium methoxidemediated ring closure.

Example 17, Step A4-Amino-5-[(E/Z)-1-cyano-2-ethenyl]-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(17.1)

To a solution of 11.2 (200 mg, 0.49 mmol) in DMF (5 mL) were addedcopper iodide (19 mg, 0.1 mmol), acrylonitrile (0.65 mL, 9.8 mmol),triethylamine (0.137 mL, 0.99 mmol), andtetrakis(triphenylphosphine)palladium [0] (57 mg, 0.05 mmol). Theresulting mixture was stirred at 70° C. for 4 d., then cooled to roomtemperature, diluted with MeOH/CH₂Cl₂ (1/1, 6 mL), and treated withDowex HCO₃ ⁻ (0.5 g). After 1 hr stirring the resin was filtered off,washed with CH₂Cl₂/MeOH (1/1, 50 mL) and combined filtrate concentrated.The crude residue was purified on a silica gel column with a CH₂Cl₂/MeOHgradient (50/1, 30/1, 10/1) to yield the target stereoisomeric mixture(B/Z, 3/1) as yellow solid (75 mg, 46%).

Example 17, Step B7-Amino-2-(2-C-methyl-β-D-ribofuranosyl)-2H-2,3,5,6-tetraazabenzo[cd]azulene(17.2)

A mixture of the compound from Step A (75 mg, 0.23 mmol) in 0.1 NNaOMe/MeOH (18 mL) was heated at 60° C. for 8 hr then cooled to roomtemperature and evaporated in vacuo. Crude residue was purified on asilica gel column with a CH₂Cl₂/MeOH gradient (20/1, 10/1, 5/1) to yieldthe target compound 17.2 as yellow solid (44 mg, 59%).

¹H-NMR (DMSO-d₆): δ 8.10 (s, H-4, 1H), 7.51 (s, 1H, H-1), 7.6-8.0 (2br,NH₂, 2H), 6.85 (d, H-9, J_(H8,H9)=11.6 Hz, 1H), 6.01 (s, H-1′, 1H), 5.52(d, H-8, J_(H8, H9), 11.6 Hz, 1H), 5.11 (m, 2′-OH, 3′-OH, 5′-OH),3.77-3.91 (m, H-3′, H-4′, H-5′, 3H), 3.63 (m, H-5′,1H), 0.70 (s, Me,3H). MS m/z=390.8 (M+CH₃COO⁻).

Example 187-Methoxy-2-(2-C-methyl-β-D-ribofuranosyl)-2H-2,3,5,6-tetraazabenzo[cd]azulene(18.3)

Peracetylated nucleoside 18.1, prepared by treating the compound 6.6with acetic anhydride, triethylamine and DMAP in acetonitrile and wasreacted with trimethyloxonium tetrafluoroborate in CH₂Cl₂ at ambienttemperature to furnish methoxy nucleoside 18.2. Removal of acetyl groupsin MeOH saturated with potassium carbonate yielded the target nucleoside18.3.

Example 18, Step A2-(2,3,5-Tri-O-acetyl-2-C-methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulene-7-one(18.1).

To a solution of2-(2-C-methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulene-7-one6.6 (136 mg, 0.41 mmol) in acetonitrile were added acetic anhydride(0.71 mL, 7.5 mmol), triethylamine (1.05 mL), and DMAP (58 mg, 0.47mmol). The mixture was stirred overnight at room temperature, thenevaporated and the residue partitioned between water (75 mL) and CH₂Cl₂(200 mL). The organic layer was washed with brine and dried over Na₂SO₄.The evaporated residue was treated with MeOH to yield the targetcompound as yellow solid (150 mg, 80%).

Example 18, Step B7-Methoxy-2-(2,3,5-tri-O-acetyl-2-C-methyl-β-D-ribofuranosyl)-2H-2,3,5,6-tetraazabenzo[cd]azulene-7-one(18.2).

A solution of the compound from Step A (50 mg, 0.11 mmol) in CH₂Cl₂ (1mL) and trimethoxyoxonium tetrafluoroborate (18 mg, 0.12 mmol) underargon was stirred at room temperature for 2 d. At this point thereaction was quenched with sat. aq. K₂CO₃ (1 mL) and the resultingmixture diluted with CH₂Cl₂ (50 mL), washed with water, brine and driedover Na₂SO₄. The evaporated residue was purified on a silica gel columnwith CH₂Cl₂/MeOH (50/1) as the eluent to yield the target compound 18.2as yellow solid (29 mg, 56%).

Example 18, Step C7-Methoxy-2-(2-C-methyl-β-D-ribofuranosyl)-2H-2,3,5,6-tetraazabenzo[cd]azulene(18.3).

A mixture of the compound from Step B (28 mg, 0.06 mmol) in saturatedmethanolic K₂CO₃ (5 mL) was stirred at room temperature for 30 min. andthen concentrated in vacuo. The crude evaporated residue was purified ona silica gel column with CH₂Cl₂/MeOH (20/1) as the eluent to afford thetarget compound 18.3 (15 mg, 72%) as a yellow solid.

¹H-NMR (DMSO-d₆): δ 8.30 (s, H-4, 1H), 7.68 (s, 1H, H-1), 6.83 (d, H-9,J_(H8,H9)11.6 Hz, 1H), 5.98 (s, H-1′, 1H), 5.84 (d, H-8, J_(H8,H9)11.4Hz, 1H), 5.22 (s, 2′-OH, 1H), 5.17 (m, 3′-OH, 1H), 5.12 (t, 5′-OH,J_(5′OH,H5′) 5.0 Hz, 1H), 3.78-3.88 (m, H-3′, H-4′, H-5′, 3H), 3.65 (m,H-5′,1H), 3.49 (s, OMe, 3H), 0.72 (s, Me, 3H). MS m/z=405.9 (M+CH₃COO⁻).

Example 192-(2-C-Methyl-β-D-ribofuranosyl)-2H-2,3,5,6-tetraazabenzo[cd]azulene-4,7(3H,6H)-dione(19.1)

To a solution4-amino-2-(2-C-methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulene-7-one15.6 (35 mg, 0.1 mmol) in 50% aqueous acetic acid (5 mL) was addedsodium nitrite (42 mg, 0.6 mmol) and the mixture stirred at roomtemperature for 4 hr. The mixture was neutralized with 1M TEAB bufferand purified by reversed phase ion-pairing HPLC on a Phenomenex LunaC18(2) 250×21 mm 10 μm column. 100 mM triethylammonium acetate (TEAA),pH 7 was used as the ion-pairing agent. A gradient of 20% to 55% MeOHover 40 min was applied. The target compound eluted at 26 min followedby two smaller peaks at 29 and 31 min. TEAA was removed by repeatedlyophilzation to yield the target compound as a fluffy yellow material(28 mg, 80%).

¹H-NMR (DMSO-d₆): δ 11.0 (br s, 2NH, 2H), 7.53 (s, 1H, H-1), 6.95 (d,H-9, J_(H8,H9) 11.7 Hz, 1H), 5.91 (s, H-1′, 1H), 5.62 (d, J_(H8,H9) 11.7Hz, H-8, 1H), 5.10 (br, 5′-OH, 3′-OH, 2′-OH, 3H), 3.77-3.89 (m, H-3′,H-4′, H-5′, 3H), 3.63 (m, H-5′,1H), 0.79(s, Me, 3H). MS m/z 347.7 (M−1).

Example 204-Chloro-2-(2-C-methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(20.8)

The tricyclic nucleoside 20.8 was synthesized starting from2-amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine 20.1. The nucleobase 20.1was diazotized in the presence of copper chloride and the resulting base2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine was treated withN-iodosuccinimide in THF at room temperature to provide2,4-dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine 20.3. Then 20.3 wasconverted to the corresponding sodium salt with sodium hydride inacetonitrile and reacted with bromo-sugar 2.4, which was prepared from3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-1-O-methyl-α-D-ribofuranose((i)Helv. Chim. Acta. 1995, 78, 486; (ii) WO 02/057287, 2002), to givenucleoside 20.4. The glycosylation product 20.4 was allowed to reactwith ammonium hydroxide in dioxane at 100° C. provided 20.5.Methyl-cis-β-(tributylstannyl)acrylate (J. Am. Chem. Soc; 1993, 115,1619) was coupled with compound 20.5 under Stille reaction conditionsusing PdCl₂(PPh₃)₂ and copper iodide to give Z-ester analog 20.6, whichwas further reacted with DBU in dioxane to give protected tricycle 20.7.Nucleoside 20.7 was treated with boron trichloride in CH₂Cl₂ to affordtricyclic nucleoside 20.8.

Example 20, Step A 2-Amino-4-chloro-5-iodo-1H-pyrrolo[2,3-d]pyrimidine(20.2)

Compound 20.2 was prepared as described in Seela, F., at al., LiebigsAnn. Chem., 1985, 312-320.

Example 20, Step B 2,4-Dichloro-5-iodo-1H-pyrrolo[2,3-d]pyrimidine(20.3)

Compound 20.2 (3.80 g, 20.0 mmol) was dissolved in THF (200 mL) andcooled to −20° C. for 20 min. N-Iodosuccinimide (7.0 g, 30.0 mmol) wasslowly added and the resulting mixture was stirred at room temperature.After 2 h, the mixture was evaporated to dryness and the residue wasre-dissolved in ethyl acetate, washed with 5% sodium thiosulphate,saturated sodium chloride solution and then dried over sodium sulfateand evaporated to dryness. The crude product was purified by silica gelcolumn chromatography using 20% ethyl acetate in hexane to give 4.6 g ofcompound 20.3 as a yellowish solid.

Example 20, Step C2,4,-Dichloro-5-iodo-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(20.4)

A solution of 2.4 ((i) Helv. Chim. Acta. 1995, 78, 486; (ii) WO02/057287, 2002) (8.8 g, 20.0 mmoles) in anhydrous acetonitrile (300 mL)was added to a solution of the sodium salt of4,6-dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine [generated in situ from4,6-dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (3.1 g, 10.0 mmol) inanhydrous acetonitrile (100 mL), and NaH (60% in mineral oil, 0.90 g,37.0 mmol), after 4 hr of vigorous stirring at room temperature]. Thecombined mixture was stirred at room temperature for 40 hr, and thenevaporated to dryness. The mixture was filtered through a celite plugand the solid residue was thoroughly washed with 500 mL of acetonitrile.The filtrates were evaporated to dryness and the crude product waspurified on a silica gel column using 25% ethyl acetate in hexane togive 2.8 g of the desired product 20.4 as a white foam.

Example 20, Step D4,2-Dichloro-5-iodo-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(20.5)

The material from Step C (2.5 g, 2.5 mmol) in dioxane (50 mL) was placedin a pressure vessel and aqueous ammonium hydroxide (50 mL) was added.The mixture was tightly sealed and heated to 100° C. for 2 hr. After thereaction, the mixture was evaporated to dryness and the crude productwas purified using silica gel column chromatography with 5-10% MeOH inCH₂Cl₂ as eluent to give 2.10 g of the pure desired product 20.5.

Example 20, Step E4-Amino-2-chloro-5-[1-(methoxycarbonyl)-2-ethenyl]-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(20.6).

To the material from Step D (2.1 g, 1.90 mmol) in 100 mL of anhydrousDMF was added CuI (83.80 mg, 0.44 mmol),methyl-cis-β-(tributylstannyl)acrylate (1.5 mL, 4.4 mmol) andPdCl₂(PPh₃)₂ (150.0 mg, 0.22 mmol) at room temperature under an argonatmosphere. The reaction mixture was heated at 70° C. for 24 hr thencooled to room temperature and filtered through a celite plug. Thefiltrate was evaporated to dryness and the crude product purified on asilica gel column using 5-30% THF in CH₂Cl₂ as eluent to give 2.2 g ofpure the desired ester 20.6.

Example 20, Step F2-[3,5-Bis-O-(2,4-dichlorophenylmethyl)2-C-methyl-β-D-ribofuranosyl]-4-chloro-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(20.7)

To a solution of compound 20.6 (1.9 g, 2.5 mmol) in dioxane (40 mL) wasadded DBU (1.3 mL, 9.0 mmol). The mixture was heated at reflux for 2 hrand then evaporated to dryness. The crude product was purified by silicagel column chromatography using 5-10% MeOH in CH₂Cl₂ as eluent to give1.7 g of pure product 20.7.

Example 20, Step G4-Chloro-2-(2-C-methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(20.8)

To a solution of compound 20.7 obtained from Step F (1.7 mg, 2.4 mmol)in CH₂Cl₂ (200 mL) at −78° C. was added boron trichloride (1M in CH₂Cl₂,25 mL, 25.0 mmol), dropwise. The mixture was stirred at −78° C. for 2.5hr, then at −30° C. to −20° C. for 3 hr. The reaction was quenched byaddition of MeOHic/CH₂Cl₂ (1:1) (50 mL) and the resulting mixturestirred at −15° C. for 30 minutes, then neutralized with aqueous ammoniaat 0° C. and stirred at room temperature for 15 minutes. The mixture wasevaporated to dryness and the residue was purified by silica gel columnchromatography using 5-20% ethanol in CH₂Cl₂ as eluent to give 560 mg ofpure yellowish tricyclic product 20.8.

¹H NMR (DMSO-d₆) δ 11.04 (d, J 1.5 Hz, 1H, NH), 7.89 (s, 1H, H-6), 7.04(d, J 11.7 Hz, H1″), 5.96 (s, 1H, H-1′), 5.69 (dd, J 11.7, 1,5 Hz, H2″),5.16 (m, 3H, 3×OH), 3.88-3.32 (m, 4H, H-3′, H-4′, 2×H-5′), 0.76 (s, 3H,CH₃).

Tricyclic nucleoside 20.8 was found to be a suitable intermediate forthe synthesis of C-4-functionalized tricyclic nucleosides. Nucleoside20.8 was reacted with sodium thiomethoxide in DMF at elevatedtemperature. Two products were isolated in a ratio of 1:1. Theseproducts were separated using reverse phase HPLC and characterized astricyclic nucleosides 21.1 and 21.2 using ¹H NMR and LCMS analysis.Compound 20.8 was treated with tert-butyldimethylsilyl chloride andimidazole in DMF at room temperature to give 5′-TBDMS protected tricylicnucleoside 21.3. Nucleophilic displacement reaction of compound 21.3using 2M methylamine in THF, followed by deprotection reaction usingtetrabutylammoniumfluoride (TBAF) afforded 4-methylamino derivative21.4. In a similar fashion, 21.3 was reacted with sodium methoxide inMeOH at reflux and the resulting product was deprotected with TBAF togive 4-OMe analog 21.5.

Examples 21 and 222-(2-C-methyl-β-D-ribofuranosyl)-4-methylthio-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(21.1) and2-(2-C-methyl-β-D-ribofuranosyl)-4,9-di(methylthio)-2,6,8,9-tetrahydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(21.2)

To a solution of the compound obtained from Example 20 (20.8) (100.0 mg,0.30 mmol) in DMF (10 mL) was added sodium thiomethoxide (105 mg, 1.5mmol). The mixture was stirred at 120° C. for 24 hr. After evaporationof the DMF under reduced pressure, the residue was purified on a silicagel column using 5-12% MeOH in CHCl₃ to give a mixture of two products(1:1). These two products were separated by reverse phase HPLC to give25 mg of 21.1 and 30 mg of 21.2.

For Example 21 (21.1): ¹H NMR (CD₃OD) δ 7.70 (s, 1H, H-6), 7.00 (d, J12.0 Hz, H1″), 6.19 (s, 1H, H-1′), 5.72 (d, J 12.0 Hz, H2″), 4.07-3.80(m, 4H, H-3′, H-4′, 2×H-5′), 2.56 (s, 3H, SCH₃), 0.91 (s, 3H, CH₃).

For Example 22 (21.2): ¹H NMR (CD₃OD) δ 7.54 (s, 1H, H-6), 6.29 (s, 1H,2×H1′), 4.51-3.79 (m, 5H, H-3′, H-4′, 2×H-5′, CH), 3.21 (m, 2H, CH₂),2.59 (s, 3H, SCH₃), 2.14 (2×s, 3H, SCH₃), 0.88 (2×s, 3H, 2×CH₃).

Example 23 2-(2-C-M4-β-D-ribofuranosyl)-4-methylamino-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(21.4)4-Chloro-2-(5-O-tert-butyldimethylsiliyl-2-C-methyl-β-D-ribofuranosyl)-2,6-dihdro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(21.3)

To a solution of compound 20.8 (366 mg, 1.0 mmol) and imidazole (68.0mg, 1.0 mmol) in DMF (20 mL) was added tert-butyldimethylsilylchloride(150.7 mg, 1.0 mmol). The mixture was stirred at ambient temperature for6 hr under inert atmosphere and then treated with saturated sodiumbicarbonate solution and extracted with ethyl acetate, dried over sodiumsulfate, and evaporated to dryness. The crude product was purified usingsilica gel column chromatography using 2-5% MeOH in CHCl₃ to give 380 mgof the desired product 21.3.

¹H NMR (CDCl₃) δ 7.68 (s, 1H, H-6), 7.24 (d, J 11.4 Hz, H1″), 6.83 (s,1H, H-1′), 5.85 (d, J 11.4 Hz, H2″), 4.14-3.72 (m, 4H, H-3′, H-4′,2×H-5′), 0.99 (s, 12H, CH₃, (CH₃)₃), 0.18 (s, 6H, 2×CH₃)

2-(2-C-Methyl-β-D-ribofuranosyl)-methylamino-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one(21.4)

Compound 21.3 (100 mg, 0.20 mmol) was added to 2M methylamine solutionin THF (25 mL) in a pressure vessel. The vessel was tightly sealed andheated at 90° C. for 8 hr. After evaporation of the solvent and excessamine, the residue was re-dissolved in THF (20 mL). To this solution,tetrabutylammonium fluoride in THF (2 mL) was added and the solutionstirred at room temperature for 4 hr. After careful evaporation of thesolvent, the residue was purified on a silica gel column using 5-7% MeOHin CHCl₃ as eluent to give 46 mg of desired yellowish product.

¹H NMR (DMSO-d₆) δ 10.05 (br s. 1H, NM), 7.42 (s, 1H, H-6), 6.89 (d, J11.7 Hz, H1″), 6.65 (br s, 1H, NH), 5.93 (s, 1H, H-1′), 5.61 (d, J 11.7Hz, H2″), 5.19-5.00 (m, 3H, 3×OH), 3.90-3.3.63 (m, 4H, H-3′, H-4′,2×H-5′), 3.32, (s, 3H, NCH₃), 0.80 (s, 3H, CH₃).

Example 24 4-Methoxy-2-(2-C-methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one (21.5)

To a solution of compound 21.3 (100.0 mg, 0.20 mmol) in anhydrous MeOH(25 mL) was added freshly prepared sodium methoxide (540.0 mg, 10.0mmol). The resulting homogeneous solution was heated to reflux for 24 hrand then neutralized with DOWEX H⁺ resin and filtered. The neutralmethanolic solution was evaporated and the residue was re-dissolved inTHF. To this solution, tetrabutylammonium fluoride in THF (2 mL) wasadded and the mixture was stirred at room temperature for 4 hr. Aftercareful evaporation of the solvent, the residue was purified on a silicagel column to give 38 mg of the desired yellowish product

¹H NMR (CD₃OD) δ 7.64 (s, 1H, H-2), 7.00 (d, J 11.7 Hz, H1″), 6.129 (s,1H, H-1′), 5.73 (d, J 11.7 Hz, H2″), 3.81-4.13 (m, 7H, H-3′, H-4′,2×H5′, OCH₃), 0.92 (s, 3H, CH₃).

Example 252-(2-C-Methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulene-7-thione(23.2)

Example 25, Step A2-(2-C-Methyl-2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulene-7-thione(23.1).

Compound 18.1 (250 mg, 0.54 mmol) was dissolved in dioxane (5 mL) andpyridine (7 mL) was added, followed by phosphorus pentasulfide (242 mg,0.5 mmol). The mixture was heated at reflux for 24 hr then the solventwas evaporated and the residue was washed with pyridine (3×4 mL). Thecombined washings were evaporated and the residue was dissolved in 50 mLCHCl₃ and washed with 30 mL 10% aqueous sodium bicarbonate followed bywater. The organic phase was dried over anhydrous sodium sulfate,filtered, evaporated and the residue (180 mg) containing 23.1 was usedimmediately in the next step.

Example 25, Step B2-(2-C-Methyl-β-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulene-7-thione(23.2).

To a suspension of compound 23.1 (180 mg, 0.38 mmol) in 4 mL ethanol wasadded 0.22 mL of 1N sodium hydroxide solution in water. The mixture wasstirred at room temperature for 1.5 hr. after which time the pH wasbrought to 6 with acetic acid and the solvent evaporated. The residuewas purified on silica gel column (10:1 CH₂Cl₂:MeOH) to afford 30 mg ofproduct 23.2. ¹H NMR (DMSO-d₆) δ 10.49 (s, 1H), 8.52 (s, 1H), 7.50 (s,1H), 7.00 (d, J 12 Hz, 1H), 6.08 (s, 1H), 5.65 (d, J 12 Hz, 1H), 5.18(m, 3H), 3.82 (m, 3H), 3.68 (m, 1H), 0.73 (s, 3H).

Example 262-(2-C-Methyl-β-D-ribofuranosyl)-6,7-dihydro-2H-2,3,5,6-tetraazabenzo[cd]azulene(24.2)

Example 27, Step A2-[3,5-Bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-β-D-ribofuranosyl]-6,7-dihydro-2H-2,3,5,6-tetraazabenzo[cd]azulene(24.1).

To compound 6.2 (372 mg, 0.5 mmol) and tri-n-butyltin allylamine(prepared according to the literature procedure in Corriu et. al.Journal of Organic Chemistry 1993, 58, 1443-1448) in 10 mL anhydroustoluene was added tetrakis(triphenylphosphine) palladium [0] and themixture was heated at reflux for 5 hr. The solvent was evaporated andthe residue dissolved in CH₂Cl₂ and loaded on a silica gel column andeluted successively with 75:1, 60:1, 40:1 CH₂Cl₂:MeOH. Pooling andevaporation of the fractions afforded 160 mg of product 24.1.

Example 27, Step B2-(2-C-Methyl-β-D-ribofuranosyl)-6,7-dihydro-2H-2,3,5,6-tetraazabenzo[cd]azulene(24.2)

To compound 24.1 (160 mg, 0.25 mmol) in CH₂Cl₂ (10 mL) at −78° C. wasadded 1M solution of boron trichloride in CH₂Cl₂ dropwise over 5 minutesand the solution stirred at −78° C. for 2.5 h and then at −25° C. for 3hr. To this mixture was added 25 mL of 1:1 v/v CH₂Cl₂:MeOH and thesolution stirred at −15° C. for 30 minutes. The mixture was brought toroom temperature and the solvent was evaporated under reduced pressure.The residue was co-evaporated with MeOH (5×10 mL) and a 10 mL MeOHsolution was neutralized with NH₄OH and evaporated again. The residuewas adsorbed on 2 g silica gel and loaded on a silica column and elutedsuccessively with 50:1,20:1 and 15:1 CH₂Cl₂:MeOH. Fractions eluting at20:1 and 15:1 were collected, Pooling of fraction and evaporation gave16 mg product 24.2. ¹H NMR (DMSO-d₆) δ 8.10 (s, 1H), 7.51 (s, 1H), 7.30(t, 1H), 6.61 (d, J 9 Hz, 1H), 6.11 (s, 1H), 5.71 (dt, J 6, 12 Hz, 1H),5.08 (m, 3H), 3.86 (m, 5H), 3.78 (m, 1H), 0.72 (s, 31).

Example 279-Methoxy-2-(2-C-methyl-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene(25.1).

The slower moving fi-action from Step B in Example 27, Step B wasisolated to afford 8 mg of ether 25.1. ¹H NMR (DMSO-d₆) δ 8.00 (s, 1H),7.53 (m, 3H), 6.11 (s, 1H), 5.11 (m, 4H), 4.43 (m, 1H), 3.97 (m, 1H),3.84 (m, 3H), 3.66 (m, 2H), 3.26 (s, 3H), 0.68(s, 3H).

Example 28 Nucleoside Monophosphates

To the compound appropriate nucleoside (0.156 mmol) (dried over P₂O₅ invacuo overnight) was added trimethyl phosphate (1.5 mL). The mixture wasstirred overnight in a sealed container containing 4A molecular sieves.It was then cooled to 0° C. and phosphorous oxychloride (35.8 μL, 2.5eq.) was added via syringe. The mixture was stirred for 3 hr at 0° C.,then the reaction was quenched by addition of tetraethylammoniumbicarbonate (TEAB) (1M) (1.5 mL) and water (15 mL). The aqueous solutionwas washed with CHCl₃ and ether then lyophilized. The crude product waspurified by HPLC using a C18 column with water and 5% acetonitrile inwater to provide the monophosphate as a triethylammonium salt afterlyophilization.

Example 29 5′-p-Phenyl Methoxyalaninylphosphate Prodrugs

To a solution of compound the appropriate nucleoside (0.6 mmol) inanhydrous THF (5 mL) was added phenyl methoxyalaninylphosphorochloridate(40 mg, 5 eq.) (freshly prepared following the literature procedure: J.Med. Chem. 1993, 36, 1048-1052 and Antiviral Research, 1999, 43, 37-53)and 1-methylimidazole (95 μL, 10 eq.) at room temperature under argon.The reaction was followed by TLC. After 36 hr, the reaction mixture wasevaporated and the residue was purified on silica gel with 0-10% MeOH inCH₂Cl₂ as eluent to provide a 1:1 mixture of diastereomers.

Example 30 2-[5-O-Bis(pivaloyloxymethyl)phoshoryl-prodrugs

To a solution of triethylammonium salt of compound nucleosidemonophosphate (0.024 mmol) in anhydrous MeOH (0.5 mL) was addedtributylstannyl methoxide (14 μL, 2 eq.) at room temperature underargon. The reaction mixture was stirred at room temperature for 30 minthen evaporated and co-evaporated with acetonitrile three times. Theresidue was dissolved in anhydrous acetonitrile (3 mL) andtetrabutylammonium bromide (15.5 mg, 2 eq.) and iodomethyl piovalate (58mg, 10 eq) were added. The reaction mixture was heated at reflux for 1hr cooled to room temperature and the solvent was evaporated. Theresidue was purified on a silica gel column with 1-5% MeOH in CH₂Cl₂ toprovide the prodrug.

Example 31 Nucleoside Diphosphates

To a solution of the triethylammonium salt of 5′-monophosphate (0.031mmol) [dried by coevaporation with anhydrous DMF twice (2×1 mL)] in 0.5mL of anhydrous DMF was added N,N′-carbonyldiimidazole (25 mg, 5 eq.) atroom temperature under argon. The reaction mixture was stirred at roomtemperature for 4 hr after which analytical TLC showed no startingmaterial. Then tributylammonium phosphate salt (1.5 n-Bu₃N/phosphate,which was prepared (see PCT, WO 88/03921) and further dried bycoevaporation with anhydrous DMF three times) was added to the abovesolution. The reaction was followed by TLC and typically after 3 days,LC-MS showed significant (>50%) conversion to product. The reaction wasquenched with 1 mL of triethylamine, 1 mL of water, and stirred at roomtemperature for 40 min. The crude product was purified by reverse phaseHPLC to provide pure product 29.1.

Examples 32-42 Nucleoside 5′-triphosphates

To an ice-cold mixture of nucleoside (0.1 mmol) in trimethyl phosphate(1 mL, anhydrous) was added POCl₃ (18.6 μL, 0.2 mmol) and the mixturestirred at 0° C. for 1 h. ributyl amine (71.5 μL, 0.3 mmol) was added,followed by acetonitrile (0.1 mL, anhydrous) and tributylammoniumpyrophosphate (182 mg, 0.4 mmol). After 30 min. the reaction wasquenched with ice-cold 1M triethylammonium bicarbonate buffer (5 mL, 1M,pH 8.5). The products were purified by HPLC. Calculated Observed ExampleMolecular m/z Number Structure Weight [M − H]⁻ 32

572.251 571.6 33

573.239 572.0 34

562.26 561.9 35

560.284 559.9 36

572.251 571.8 37

587.266 586.9 38

574.267 573.9 39

542.225 541.1 40

590.241 589.8 41

586.278 585.8 42

602.277 601.9

Example 43 Nucleoside-5′triphosphate Mimic2-(5-α-P_(I)-borano-β,γ-difluoromethylene)triphoshono2-C-methyl-β-D-ribofuranosyl)-2,6,8,9-tetrahydro-7-oxa-2,3,5,6-tetraazabenzo[cd]azulene(43.2)

Example 43, step A2-(2-C-methyl-2,30di-O-acetyl-β-D-ribofuranosyl)-2,6,8,9-tetrahydro-7-oxa-2,3,5,6-tetraazabenzo[cd]azulene(43.1)

To a solution of compound 2.9 from example 2, step G (76 mg, 0.24 mmol)in pyridine (2.0 mL) was added tert-butyldimethylsilylchloride (60 mg,0.38 mmol). The mixture was stirred at ambient temperature for 16 hrunder inert atmosphere. Added acetic anhydride (0.44 mL, 4.32 mmol), nadstirred for 3 h at room temperature. Added triethylamine (0.61 mL, 6.0mmol), and DMAP (35 mg, 0.29 mmol). The mixture was stirred overnight atroom temperature, then evaporated and the residue partitioned betweenwater (20 mL) and CH₂Cl₂ (60 mL). The organic layer was dried overNa₂SO₄. Solvents were evaporated in vacuo. The resulted crude productwas dried on high vacuum for 2 h and redissolved in THF (2.0 ml) andcooled to 0° C. and added 1.0M solution of TBAF in THF (0.6 mmol) andstirred at 0° C. for 1 h. Reaction was quenched by adding absoluteethanol (2 ml), and solvents were evaporated. The residue left wasredissolved in dichloromethane and washed with water. The organicportions were dried over Na₂SO₄ and filtered, evaporated and crudeproduct was purified by silicagel column chromatography using 1-2%methanol in dichloromethane to give 43.1 as light yellow foam (57 mg).

¹H NMR (DMSO-d₆) d 8.57 (s, H-2, 1H), 7.70 (s, H-6, 1H), 6.62 (s, H-1′,1H), 5.39 (d J 5.7, H-3′, 1H), 5.23 (t, J 5.7, 5′-OH, 1H), 4.52 (br s,OCH₂CH₂, 2H), 4.09-4.13 (m, H-4′, 1H), 3.62-3.81 (m, H-5′, 2H), 3.08-3.1(m, OCH₂CH₂, 2H), 2.51 (s, N—COCH₃, 3H), 2.08, 2.04 ( each s, 2×O—COCH₃,6H), 1.34 (s, CH₃, 3H).

Example 43, Step B 2-(5-α-P_(I)-borano-β,γ-difluoromethylene)triphoshono2-C-methyl-β-D-ribofuranosyl)-2,6,8,9-tetrahydro-7-oxa-2,3,5,6-tetraazabenzo[cd]azulene(43.2)

2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one (29 mg, 0.15 mmol) wasadded to a stirred solution of 43.1 (43 mg, 0.1 mmol) in anhydrous DMF(0.5 mL) and pyridine (0.1 mL) at 0° C. under argon. The reactionmixture was stirred at room temperature for 2 h, cooled with ice bath.Tributylamine (65 uL, 0.28 mmol) was added, followed by addition of(difluoromethylene)diphosphonic acid bis(tributylammonium) salt (89 mg,0.15 mmol) in DMF (0.5 mL). The reaction mixture was stirred at roomtemperature for 2 h and cooled with ice. Borane-diisopropylethylaminecomplex (377 ul, 2.11 mmol) was added, and the resulting mixture wasstirred at room temperature for 6 h, cooled with ice, and quenched byslow addition of water (2 mL). The mixture was stirred at roomtemperature for 1 h, diluted with water (3 ml), extracted two times withchloroform and aqueous portion was concentrated to about 2 ml. Aqueousammonia (33%) (2 ml) was added and stirred at room temperature for 10 hand ammonia was evaporated and the remaining aqueous portion wasanalysed by LCMS. LCMS showed the presence of two diastereoisomers of43.2. MS m/z 592.1 [M-H]

Example 44 HCV Replicon Assays

Doubling or ½-log dilutions of each compound were made in DMSO, andaliquots were transferred to 96-well microplates to give a finalconcentration range of 100-400 μM downwards in the presence of aconstant concentration of 1% DMSO (ELISA and Reporter methods) or 0.4%DMSO (hybridization method). The inhibitory activity of these wasassessed by three methods in Huh-7 cells transfected with repliconscoding for non-structural (NS) proteins of HCV.

Replicon ELISA method: Huh-7 cells containing an HCV NS3-NS5b repliconwere seeded into microplates containing compound dilutions at aconcentration of 20,000 cells per well. After 3 days incubation the cellmonolayers were washed and fixed with 1:1 acetone/MeOH. An ELISA wasperformed on the fixed cell sheets by the sequential addition ofHCV-specific monoclonal antibody, horseradish peroxidase-conjugatedsecondary antibody and substrate solution, with thorough washing betweenadditions. The colour development reaction was stopped with 12.5%sulphuric acid and the plates read at 490 nm. The monolayers were thenwashed, dried and stained with carbol fuchsin for microscopic assessmentof cytotoxicity.

Replicon reporter method: Huh-7 cells containing a replicon expressingHCV NS3-NS5b plus a reporter gene were seeded into the test microplatesat a concentration of 15,000 cells per well. After 2 days incubation,the viability of the cells was assessed by the addition of Resazurin(Sigma TOX-8) to all wells and reading the plates at 595 nm 3 hours postaddition. The signal from the reporter gene product was measuredimmediately thereafter.

Replicon hybridization and cytotoxicity method: Huh-7 cells containingan HCV NS3-NS5b replicon were seeded into microplates containingcompound dilutions at a concentration of 5,000 cells per well. After 3days incubation the media was replaced with MTS solution andcytotoxicity was assessed by color development. After reading the platesat 490 nm, the MTS solution was aspirated and the cells were lysed andhybridized against HCV sequences using a chemiluminescent readout.

Data analysis: The mean reading of duplicate wells at each compoundconcentration was expressed as a percentage of the mean value forcompound-free control wells. Percentage inhibition was plotted againstconcentration for each compound, and the 50% inhibitory concentration(IC₅₀) was calculated.

Compounds of Examples 1-27 were typically active in replicon assays inthe range of 5 to >1000 μM

Example 45

Huh-7 and Vero Cells: The compounds were additionally assessed forcytotoxicity in exponentially growing Huh-7 and Vero cell cultures.Doubling dilutions of the compounds were made, as described previously,and transferred to test microplates to give final concentrations of 500μM downwards. Either Huh-7 cells or Vero cells were added atconcentrations of 12,000 and 3,000 cells per well respectively. Afterincubation for 4 days at 37° C., MTT was added to all wells and theplates re-incubated for 2 hours. Acidified isopropanol was then added toall wells to lyse the cells and dissolve any formazan that had beenproduced. Absorbance was read at 570 nm, and the mean readings fromduplicate test wells were expressed as percentages of the mean readingsfrom compound free control wells. The 50% cytotoxic concentration(CCID₅₀) of each compound was calculated from the plot of percentagecell survival against compound concentration.

HFF Cells: Cells were seeded into microtiter plates containing ½-logdilutions of compounds at a concentration of 5,000 cells per well. After3 days incubation the media was replaced with MTS solution in media andcytotoxicity was assessed by color development. Plates were read at 490nm and CC₅₀s were calculated from percent inhibition as noted above.

Huh-7 and Vero Cells: The compounds were additionally assessed forcytotoxicity in exponentially growing Huh-7 and Vero cell cultures.Doubling dilutions of the compounds were made, as described previously,and transferred to test microplates to give final concentrations of 500μM downwards. Either Huh-7 cells or Vero cells were added atconcentrations of 12,000 and 3,000 cells per well respectively. Afterincubation for 4 days at 37° C., MTT was added to all wells and theplates re-incubated for 2 hours. Acidified isopropanol was then added toall wells to lyse the cells and dissolve any formazan that had beenproduced. Absorbance was read at 570 nm, and the mean readings fromduplicate test wells were expressed as percentages of the mean readingsfrom compound free control wells. The 50% cytotoxic concentration(CCID₅₀) of each compound was calculated from the plot of percentagecell survival against compound concentration.

HFF Cells: Cells were seeded into microtiter plates containing ½-logdilutions of compounds at a concentration of 5,000 cells per well. After3 days incubation the media was replaced with MTS solution in media andcytotoxicity was assessed by color development. Plates were read at 490ml and CC₅₀s were calculated from percent inhibition as noted above.

Huh-7 and Vero Cells: The compounds were additionally assessed forcytotoxicity in exponentially growing Huh-7 and Vero cell cultures.Doubling dilutions of the compounds were made, as described previously,and transferred to test microplates to give final concentrations of 500μM downwards. Either Huh-7 cells or Vero cells were added atconcentrations of 12,000 and 3,000 cells per well respectively. Afterincubation for 4 days at 37° C., MTT was added to all wells and theplates re-incubated for 2 hours. Acidified isopropanol was then added toall wells to lyse the cells and dissolve any formazan that had beenproduced. Absorbance was read at 570 nm, and the mean readings fromduplicate test wells were expressed as percentages of the mean readingsfrom compound free control wells. The 50% cytotoxic concentration(CCID₅₀) of each compound was calculated from the plot of percentagecell survival against compound concentration.

HFF Cells: Cells were seeded into microtiter plates containing ½-logdilutions of compounds at a concentration of 5,000 cells per well. After3 days incubation the media was replaced with MTS solution in media andcytotoxicity was assessed by color development. Plates were read at 490nm and CC₅₀s were calculated from percent inhibition as noted above.

Compounds of Examples 1-27 were typically cytotoxic in the range of 30to >100 μM.

Example 46 HCV Polymerase Inhibition Assay

The C-terminal his-tagged full-length HCV (Bartenschlager 1b) polymerasegene was cloned and expressed in Sf9 cells by standard procedures. Theenzyme was purified by nickel affinity chromatography followed byS-Sepharose column chromatography. Reactions contained 20 mM Tris HCl pH7.0, 5 mM Hepes pH 7.0, 90 mM NaCl, 12.5 mM MgCl₂, 2% glycerol, 0.005%Triton X-100, 1.5 mM DTT, 0.4 U/μd RNasin, 20 μg/ml RNA corresponding to696 nucleotides of the 3′ non-coding region of the HCV 1b genome, 2 μMUTP (=K_(m)), 0.02 μCi/μl ³³P-labelled UTP, a concentration equal to theK_(m) of competing NTP (20 μM ATP, 3 μM GTP, or 0.5 μM CTP), 500 μM“non-competing” NTPs, and 100 nM HCV 1b polymerase (Bartenschlager, falllength enzyme) in a total volume of 25 μl. Reactions were initiated withthe addition of enzyme and terminated after 2 hours with 5 μl 0.5 MEDTA. Stopped reactions were spotted onto either DEAE filter mats orDEAE 96-well filter plates (Millipore). Unincorporated nucleotides werewashed from the filters. The filter mat was dried and sealed in a bagtogether with 10 ml of OptiScint HiSafe scintillation fluid. Filterplates were dried, and 75 ml OptiPhase scintillation fluid was added toeach well. The remaining radioactivity was quantitated on a Wallac 1205Betaplate counter or Wallac 1240 MicroBeta plate counter.

Compounds of Examples 32-42 were typically inhibitory of NS5B in therange of 100 to >1000 nM. Selected Examples were more active anddisplayed IC₅₀ values in the range of 30 to 100 nM.

1. A compound of the formula (I) which may be a D- or L-nucleotide ornucleoside

wherein R¹, R², R^(2′), R³, R^(3′), and R⁴ are independently H, F, Cl,Br, I, OH, SH, NH₂, NHOH, NHNH₂, N₃, COOH, CN, CONH₂, C(S)NH₂, COOR, R,OR, SR, SSR, NHR, or NR₂, wherein R² or R^(2′) are not both hydrogen; Lis O, S, NH, NR, CY₂O, CY₂S, CY₂NH, CY₂, CY₂CY₂, CY₂OCY₂, CY₂SCY₂, orCY₂NHCY₂, wherein Y is H, F, Cl, Br, alkyl, alkenyl, or alkynyl, andwherein alkyl, alkenyl, and alkynyl may each optionally contain one ormore heteroatoms; R⁵ is OH, monophosphate, diphosphate, or triphosphateoptionally masked with prodrug moieties or a monophosphonate prodrug; Bis a base selected from:

wherein dashed lines (---) indicate an optional π bond; X is N, NH, orNR; each Z is independently N, N—BH₂G⁻M⁺, C-G, O, S,NR, >C═O, >C═S, >C═NH, >C═NR, >S═O, >S(O)₂ or CH-G, wherein if Z is aparticipant in a π bond then each Z is independently N or C-G, and if Zis not a participant in a π bond then each Z is independentlyN—(BH₂G)⁻M⁺, O, S, NR, >C═O, >C═S, >C═NH, >C═NR, >S═O, >S(O)₂ or CH-G;BH₂G⁻M⁺ is an ion pair and M⁺ is a cation; Z³ is CH or N; Z⁴ isC-G, >C═O, >C═S, >C═NH or >C═NR, wherein if Z⁴ is a participant in a πbond then Z⁴ is C-G, and if Z⁴ is not a participant in a π bond then Z⁴is >C═O, >C═S, >C═NH or >C═NR; each G is independently H, F, Cl, Br, I,OH, SH, NH₂, NHOH, N₃, COOH, CN, CONH₂, C(S)NH₂, C(═NH)NH₂, R, OR, SR,NHR, or NR₂; and each R is independently alkyl, alkenyl, alkynyl, aryl,acyl, or aralkyl, optionally containing one or more heteroatoms; or apharmaceutically acceptable salt thereof.
 2. The compound of claim 1wherein Z⁴ is a participant in a π bond, and base B is:


3. The compound of claim 2 wherein G is H, F, Cl, Br, I, R, OR, SR, NH₂,NHR or NR₂, and R is alkyl.
 4. The compound of claim 2 wherein X is NHor NR, and base B is selected from:


5. The compound of claim 2 wherein X is N and base B is:


6. The compound of claim 1 wherein Z⁴ is not a participant in a π bond,and base B is selected from:


7. The compound of claim 6 wherein X is NH or NR, and base B is selectedfrom:


8. The compound of claim 7 wherein X is NH, and base B is selected from:


9. The compound of claim 6 wherein X is NH or NR, and base B is selectedfrom:


10. The compound of claim 6 wherein X is N, and base B is selected from:


11. The compound of claim 1 wherein the base B is selected from one ofthe following structures:


12. The compound of claim 1 wherein the nucleotide or nucleoside has thestructure


13. The compound of claim 12 wherein R¹, R³ and R⁴ are hydrogen.
 14. Thecompound of claim 13 wherein R², R^(2′) and R^(3′) are independently H,F, Cl, Br, I, OH, N₃, CN, R or OR, and R is alkyl.
 15. The compound ofclaim 13 wherein R^(2′) and R^(3′) are OH.
 16. The compound of claim 15wherein R² is H or methyl.
 17. The compound of claim 1 wherein R⁵ is OHor monophosphate, diphosphate or triphosphate optionally masked withprodrug moieties or a monophosphonate prodrug.
 18. The compound of claim1, wherein the compound is selected from one of the followingstructures:


19. A pharmaceutical composition comprising a therapeutically effectiveamount of a compound of claim 1 or a pharmaceutically acceptable saltthereof.
 20. A method for treating a viral infection comprisingadministering a therapeutically effective amount of a compound of claim1 to a mammal in need thereof, wherein the viral infection is HIV, HBVor HCV.